Immunogenic compositions comprising cold-adapted attenuated respiratory syncytial virus mutants

ABSTRACT

The respiratory syncytial virus (RSV) is a major cause of lower respiratory tract disease in infants and children throughout the world. RSV is a major cause of pneumonia and bronchiolitis in infants under one year of age, and is a major cause of fatal respiratory tract disease in these infants. The treatment and prevention of RSV infection has been problematic. However, the present invention addresses some of these concerns by providing attenuated RSV strains that are suitable for inclusion in immunizing compositions. Specifically, the present invention is directed toward the introduction of growth restriction mutations into incompletely attenuated host range-restricted cold-passaged respiratory syncytial virus (cpRSV) strains by further passage of the strains at increasingly reduced temperatures to produce derivative strains which are more satisfactorily attenuated. These cold-adaptation (ca) approaches were used to introduce further attenuation in the parental RSV virus cpRSV-3131, which is incompletely attenuated in seronegative children. Mutants of the parental strain were obtained by selecting for large plaque production at reduced temperatures. An RSV cp-3131 derivative, designated D 1 , was isolated that produces large plaques at 25° C. Biologically cloned virus D1 produces distinctly and uniformly larger plaques at 25° C. as compared to the parental attenuated strain cpRSV-3131 or wild-type strain A2. Thus, D1 is an attenuated cold-adapted, but not temperature-sensitive, RSV mutant. The invention also provides methods for stimulating RSV-specific immune responses in an individual through the administration of said mutants.

This is a divisional of application Ser. No. 08/327,263, filed on Oct.21, 1994, now U.S. Pat. No. 5,922,326 which is a continuation-in-part ofSer. No. 08/039,945, filed on Apr. 9, 1993, now abandoned, which is acontinuation-in-part of Ser. No. 07/872,746, filed Apr. 21, 1992, nowabandoned.

BACKGROUND OF THE INVENTION

Respiratory syncytial (RS) virus infection of humans ranges fromasymptomatic to severe respiratory tract disease. In infants andchildren, RS virus (RSV) is regarded as one of the most important causesof lower respiratory tract disease in all geographic areas of the world.RS virus outranks all other microbial pathogens as a cause of pneumoniaand bronchiolitis in infants under one year of age, and is a major causeof fatal respiratory tract disease in these infants. Virtually allchildren are infected by two years of age. Reinfection occurs withappreciable frequency in older children and young adults. (Chanock etal., in Viral Infections of Humans, 3rd ed., A. S. Evans, ed., PlenumPress, N.Y. (1989)). Although most healthy adults do not have seriousdisease due to RS virus infection, elderly patients andimmunocompromised individuals are more likely to have severe andpossibly life-threatening infections.

Treatment of RSV infection has been problematic. Small infants havediminished serum and secretory antibody responses to RSV antigens andthus suffer more severe infections, whereas cumulative immunity appearsto protect older children and adults against more serious forms of theinfection. One antiviral compound, ribavirin, has shown promise in thetreatment of severely infected infants, although there is no indicationthat it shortens the duration of hospitalization or diminishes theinfant's need for supportive therapy.

The mechanisms of immunity in RSV infection have recently come intofocus. Secretory antibodies appear to be most important in protectingthe upper respiratory tract, whereas high levels of serum antibodies arethought to have a major role in resistance to RSV infection in the lowerrespiratory tract. Purified human immunoglobulin containing a high titerof neutralizing antibodies to RSV may prove useful in immunotherapeuticapproaches for serious lower respiratory tract disease in infants andyoung children. Immune globulin preparations, however, suffer fromseveral disadvantages, such as the possibility of transmittingblood-bome viruses and difficulty and expense in preparation andstorage.

Despite an urgent need for an effective vaccine against RS virus,particularly for infants and young children, previous attempts todevelop a safe and effective vaccine have met with failure. Aformalin-inactivated virus vaccine tested in the mid-1960s failed toprotect against RS virus infection or disease. Instead, disease wasexacerbated during subsequent infection by RS virus. Kim et al., Am. J.Epidemiol. 89:422-434, Chin et al., Am J. Epidemiol. 89:449-463 (1969);Kapikian et al., Am. J. Epidemiol. 89:405-421 (1969).

To circumvent the problems attendant with the inactivated vaccines andthe possible alteration of neutralization epitopes, efforts weredirected to developing attenuated RS mutants. Friedewald et al., J.Amer. Med. Assoc. 204:690-694 (1968) reported the production of alow-temperature passaged mutant of RS virus which appeared to possesssufficient attenuation to be a candidate vaccine. This mutant exhibiteda slight increased efficiency of growth at 26° C. compared to itswild-type parental virus, but its replication was neither temperaturesensitive nor significantly cold-adapted. The cold-passaged mutant,however, was attenuated for adults. Although satisfactorily attenuatedand immunogenic for infants and children who had been previouslyinfected with RSV (i.e., seropositive individuals), the mutant retaineda low level virulence for the upper respiratory tract of seronegativeinfants. This RSV mutant had been passaged in bovine kidney cell cultureat low temperature (26° C.) and as a consequence it acquired host rangeattenuating mutations. The acquisition of these mutations allowed themutant to replicate efficiently in bovine tissue, whereas these samemutations restricted growth of the mutant in the human respiratory tractcompared to its RSV strain A2 parent.

Similarly, Gharpure et al., J. Virol. 3:414-421 (1969) reported theisolation of temperature sensitive (ts) mutants which also werepromising vaccine candidates. One mutant, ts-1, was evaluatedextensively in the laboratory and in volunteers. The mutant producedasymptomatic infection in adult volunteers and conferred resistance tochallenge with wild-type virus 45 days after immunization. Again, whileseropositive infants and children underwent asymptomatic infection,seronegative infants developed signs of rhinitis and other mildsymptoms. Furthermore, instability of the ts phenotype was detected,although virus exhibiting a partial or complete loss of temperaturesensitivity represented a small proportion of virus recoverable fromvaccinees, and was not associated with signs of disease other than mildrhinitis.

The aforementioned studies thus revealed that among the cold-passagedand temperature sensitive strains some were underattenuated and causedmild symptoms of disease in some vaccinees, particularly seronegativeinfants, while others were overattenuated and failed to replicatesufficiently to elicit protective immune responses. (Wright et al.,Infect. Immun. 37:397-400 (1982)). The genetic instability that allowedcandidate vaccine mutants to lose their temperature-sensitive phenotypewas also a disconcerting discovery. See generally Hodes et al., Proc.Soc. Exp. Biol. Med. 145:1158-1164 (1974), McIntosh et al. Pediatr. Res.8:689-696 (1974), and Belshe et al., J. Med. Virol. 3:101-110 (1978).

Abandoning the attenuated RS virus vaccine approach, investigatorstested potential subunit vaccine candidates using purified RS virusenvelope glycoproteins from lysates of infected cells. The glycoproteinsinduced resistance to RS virus infection in the lungs of cotton rats,Walsh et al, J. Infect. Dis. 155:1198-1204 (1987), but the antibodiesinduced had very weak neutralizing activity and immunization of rodentswith purified subunit vaccine led to disease potentiation (Murphy etal., Vaccine 8:497-502 (1990)).

Vaccinia virus recombinant-based vaccines which express the F or Genvelope glycoprotein have also been explored. These recombinantsexpress RSV glycoproteins which are indistinguishable from the authenticviral counterpart, and small rodents infected intradermally with thevaccinia-RSV F and G recombinant viruses developed high levels ofspecific antibodies that neutralized viral infectivity. Indeed,infection of cotton rats with vaccinia-F recombinants stimulated almostcomplete resistance to replication of RSV in the lower respiratory tractand significant resistance in the upper tract. Olmsted et al., Proc.Natl. Acad. Sci. USA 83:7462-7466 (1986). However, immunization ofchimpanzees with vaccinia F and vaccinia G recombinant provided almostno protection against RSV challenge in -the upper respiratory tract(Collins et al., Vaccine 8:164-168 (1990)) and inconsistent protectionin the lower respiratory tract (Crowe et al., Vaccine 11:1395-1404(1993). This led to the conclusion that this approach was not likely toyield a successful vaccine.

While investigators examined several different approaches to producingan effective and safe RS vaccine over the years, RS virus has remainedthe most common cause of severe viral lower respiratory tract disease ininfants and children. Consequently, an urgent need remains for a safevaccine that is able to prevent the serious illness in this populationthat often requires hospitalization, and to prevent disease in otherindividuals. Quite surprisingly, the present invention fulfills theseand other related needs.

SUMMARY OF THE INVENTION

The present invention provides vaccine compositions of attenuatedrespiratory syncytial virus. The attenuated virus is provided in anamount sufficient to induce an immune response in a human host, inconjunction with a physiologically acceptable carrier and may optionallyinclude an adjuvant to enhance the immune response of the host. Theinvention contemplates several distinct antigenic subgroups ofattenuated RS virus which are derived from incompletely attenuated RSvirus and which possess properties heretofore not exhibited byattenuated RS viruses previously reported in the literature. In oneembodiment thereof, the attenuated virus of the invention comprises hostrange restricted RS virus (i.e. virus possessing mutations that restrictreplication in the lung of the host) incompletely attenuated bycold-passage (cpRSV) into which at least one or more additionalmutations are introduced to produce a virus and its progeny having atemperature sensitive (ts) phenotype, which are hereinafter designatedcptsRSV. In another embodiment, host-range restricted RS virusincompletely attenuated by cold-passage (cpCRSV) is cold adapted (ca) bypassage at increasingly reduced temperatures to introduce additionalgrowth restriction mutations. In yet another embodiment, incompletelyatttenuated RSV ts mutants, such as RSV ts-4 and ts-1, NG1 are furtherattenuated by introduction of additional mutations. The attenuatedderivatives of the ts or cp strains are produced in several ways, butpreferably by introduction of additional temperature sensitive-mutationsby chemical mutagenesis, by further passage in culture at attenuatingtemperatures of 20-24° C., or by introduction of small plaque (sp)mutations and selection of derivatives which are more restricted inreplication than the incompletely attenuated parental mutant strain. Theattenuated virus of the invention belongs to either antigenic subgroup Aor B, and virus from both subgroups may conveniently be combined invaccine formulations for more comprehensive coverage against prevalentRSV infections. The vaccine will typically be formulated in a dose offrom about 10³ to 10⁶ plaque-forming units (PFU) or more for maximalefficacy.

In other embodiments, the invention provides methods for stimulating theimmune system of an individual to induce protection against respiratorysyncytial virus. These methods comprise administering to the individualan immunologically sufficient amount of RSV which has been attenuated byintroducing mutations that specify the ts, ca, and/or sp phenotype intoRSV which was originally incompletely attenuated by ts mutation(s) or bypassage at cold temperature, e.g., 26° C. In view of the potentiallyserious consequences of RSV infection in neonates, seronegative andseropositive infants and young children, and the elderly, theseindividuals will typically benefit most from imunization according tothe present methods. In this regard, seronegative individuals are thosewho exhibit no evidence of previous infection with a subgroup A or B RSvirus, while seropositive individuals can be classified as those with noprevious infection with RSV, but with RSV antibodies that have beenacquired passively from the mother, as well as those individuals whohave RSV antibodies due to past infection with RSV. A titer ofneutralizing antibody of equal to or greater than 1:20 is considered aseropositive condition. In most instances the attenuated RS virus isadministered to the respiratory tract of the individual, preferablyintranasally by aerosol or droplet application. The attenuated RSviruses of the invention have been deposited under the terms of theBudapest Treaty with the American Type Culture Collection (ATCC) of12301 Parklawn Drive, Rockville, Md. 20852, U.S.A., and were granted theaccession numbers as follows:

Virus Name ATCC Designation RSV A2 cpts - 248 VR 2450 RSV A2 cpts -530/1009 VR 2451 RSV A2 cpts - 530 VR 2452 RSV A2 cpts - 248/955 VR 2453RSV A2 cpts - 248/404 VR 2454 RSV A2 cpts - 530/1030 VR 2455

In yet other embodiments, the invention provides pure cultures ofattenuated RS virus, wherein the virus has been more completelyattenuated by the further derivatization of previously identified ts orcp mutants. The attenuated virus is capable of eliciting a protectiveimmune response in an infected human host yet is sufficiently attenuatedso as to not cause unacceptable symptoms of severe respiratory diseasein the immunized host. The attenuated virus may be present in a cellculture supernatant, isolated from the culture, or partially orcompletely purified. The virus may also be lyophilized, and can becombined with a variety of other components for storage or delivery to ahost, as desired.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph demonstrating the substantially complete correlationbetween the replication of a series of subgroup A respiratory syncytialviruses in the lungs of mice with their replication in the chimpanzee.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention provides RS virus suitable for vaccine use inhumans. The RS virus described herein is produced by introducingadditional mutations into incompletely attenuated strains of ts or cp RSvirus. The mutations are introduced into the strains during virus growthin cell cultures to which a chemical mutagen has been added, byselection of virus that has been subjected to passage at suboptimaltemperature in order to introduce growth restriction mutations, or byselection of mutagenized virus that produces small plaques in cellculture.

Thus, the vaccine of the invention comprises the attenuated RV virus anda physiologically acceptable carrier. The vaccine is administered in animmunogenically sufficient amount to an individual in need ofimmunological protection against RS virus, such as, e.g., an infant,child, the elderly, or adult candidates for immunosuppressive therapies.The vaccine elicits the production of an immune response that isprotective against serious lower respiratory tract disease, such aspneumonia and bronchiolitis when the individual is subsequently infectedwith wild-type RS virus. While the naturally circulating virus is stillcapable of causing infection, particularly in the upper respiratorytract, there is a very greatly reduced possibility of rhinitis as aresult of the vaccination and possible boosting of resistance bysubsequent infection by wild-type virus. Following vaccination, thereare detectable levels of host engendered serum and secretory antibodieswhich are capable of neutralizing homologous (of the same subgroup)wild-type virus in vitro and in vivo. In many instances the hostantibodies will also neutralize wild-type virus of a different,non-vaccine subgroup. To achieve higher levels of cross-protection,i.e., against heterologous strains of another subgroup, it is preferredto vaccinate individuals with attenuated RS virus from at least onepredominant strain of both subgroups A and B.

The attenuated virus which is a component of the vaccine is in anisolated and typically purified form By isolated is meant to refer toattenuated modified RS virus which is in other than the nativeenvironment of wild-type virus, such as the nasopharynx of an infectedindividual. More generally, isolated is meant to include the attenuatedvirus as a heterologous component of a cell culture or other system. Forexample, attenuated RS virus of the invention may be produced by aninfected cell culture, separated from the cell culture and added to astabilizer which contains other non-naturally occurring RS viruses, suchas those which are selected to be attenuated by means of resistance toneutralizing monoclonal antibodies to the F-protein, as described inco-filed U.S. patent application attorney docket 15280-11-2, thedisclosure of which is expressly incorporated herein by reference.

The attenuated RS virus of the present invention exhibits a verysubstantial diminition of virulence when compared to wild-type virusthat is circulating naturally in humans. The attenuated virus issufficiently attenuated so that symptoms of infection will not occur inmost immunized individuals. In some instances the attenuated virus maystill be capable of dissemination to unvaccinated individuals. However,its virulence is sufficiently abrogated such that severe lowerrespiratory tract infections in the vaccinated or incidental host do notoccur.

The level of attenuation may be determined by, for example, quantifyingthe amount of virus present in the respiratory tract of an immunizedhost and comparing the amount to that produced by wild-type RS virus orother attenuated RS viruses which have been evaluated as candidatevaccine strains. For example, the attenuated virus of the invention willhave a greater degree of restriction of replication in the upperrespiratory tract of a highly susceptible host, such as a chimpanzee,compared to the levels of replication of wild-type virus, e.g., 10- to1000-fold less. Also, the level of replication of the attenuated RSVvaccine strain in the upper respiratory tract of the chimpanzee shouldbe less than that of the RSV A2 ts-1 mutant, which was demonstratedpreviously to be incompletely attenuated in seronegative human infants.In order to further reduce the development of rhinorrhea, which isassociated with the replication of virus in the upper respiratory tract,an ideal vaccine candidate virus should exhibit a restricted level ofreplication in both the upper and lower respiratory tract. However, theattenuated viruses of the invention must be sufficiently infectious andimmunogenic in humans to confer protection in vaccinated individuals.Methods for determining levels of RS virus in the nasopharynx of aninfected host are well known in the literature. Specimens are obtainedby aspiration or washing out of nasopharyngeal secretions and virusquantified in tissue culture or other by laboratory procedure. See, forexample, Belshe et al., J. Med. Virology 1:157-162 (1977), Friedewald etal., J. Amer. Med. Assoc. 204:690-694 (1968); Gharpure et al., J. Virol.3:414-421 (1969); and Wright et al., Arch. Ges. Virusforsch. 41:238-247(1973). The virus can conveniently be measured in the nasopharynx ofhost animals, such as chimpanzees.

To produce a satisfactorily attenuated derivative virus of the presentinvention, mutations are introduced into a parental viral strain whichhas been incompletely or partially attenuated, such as the ts-1 or ts-4mutant, or cpRSV. For virus of subgroup A, the incompletely attenuatedparental virus is preferably ts-1 or ts-1 NG-1 or cpRSV, which aremutants of the A2 strain of subgroup A, or derivatives or subclonesthereof.

Partially attenuated mutants of the subgroup B virus can be produced bybiologically cloning wild-type subgroup B virus in an acceptable cellsubstrate and developing cold-passaged mutants thereof, subjecting thevirus to chemical mutagenesis to produce ts mutants, or selecting smallplaque mutants thereof. For virus of subgroup B, the incompletelyattenuated parental virus is preferably cp 52/2B5, which is a mutant ofthe B1 strain of subgroup B. The various selection techniques may alsobe combined to produce the partially attenuated mutants of subgroup A orB which are useful for further derivatization as described herein.

Once the desired partially attenuated parental strain(s) is/areselected, further attenuation sufficient to produce a vaccine acceptablefor use in humans according to the present invention may be accomplishedin several ways as described herein.

According to the present invention the cp mutant can be furthermutagenized in several ways. In one embodiment the procedure involvessubjecting the partially attenuated virus to passage in cell culture atprogressively lower, attenuating temperatures. For example, whereaswild-type virus is typically cultivated at about 34-37° C., thepartially attenuated mutants are produced by passage in cell cultures(e.g., primary bovine kidney cells) at suboptimal temperatures, e.g.,20-26° C. These mutants have slight but definite evidence of coldadaptation (ca), i.e., increased efficiency of growth at 26° C. comparedto its wild-type parental virus, but typically are not ts. Thus, in onemethod of the present invention the cp mutant or other partiallyattenuated strain, e.g., ts-1 sp, is adapted to efficient growth at alower temperature by passage in MRC-5 or Vero cells, down to atemperature of about 20-24° C., preferably 20-22° C. This selection ofmutant RS virus during cold-passage substantially eliminates anyresidual virulence in the derivative strains as compared to thepartially attenuated parent.

In another embodiment of the invention the incompletely attenuatedstrains are subjected to chemical mutagenesis to intrduce ts mutationsor, in the case of viruses which are already ts, additional ts mutationssufficient to confer increased stability of the ts phenotype on theattenuated derivative. Means for the introduction of ts mutations intoRS virus include replication of the virus in the presence of a mutagensuch as 5-fluorouridine or 5-fluorouracil in a concentration of about10⁻³ to 10⁵ M, preferably about 10⁴ M, or exposure of virus tonitrosoguanidine at a concentration of about 100 μg/ml, according to thegeneral procedure described in, e.g., Gharpure et al., J. Virol.3:414-421 (1969) and Richardson et al., J. Med. Virol. 3:91-100 (1978).Other chemical mutagens can also be used. Attenuation can result from ats mutation in almost any RS virus gene. The level of temperaturesensitivity of the replication of the attenuated RS virus of theinvention is determined by comparing its replication at a permissivetemperature with that at several restrictive temperatures. The lowesttemperature at which the replication of the virus is reduced 100-fold ormore in comparison with its replication at the permissive temperature istermed the shutoff temperature. In experimental animals and humans, boththe replication and virulence of RS virus correlate with the mutant'sshutoff temperature. Replication of mutants with a shutoff temperatureof 39° C. is moderately restricted, whereas mutants with a shutoff of38° C. replicate less well and symptoms of illness are mainly restrictedto the upper respiratory tract. A virus with a shutoff temperature of 35to 37° C. should be fully attenuated in humans. Thus, the attenuated RSvirus of the invention which is temperature-sensitive will have ashutoff temperature in the range of about 35 to 39° C., and preferablyfrom 35 to 38° C. The addition of the temperature sensitive property toa partially attenuated strain produces completely attenuated virususeful in the vaccine compositions of the present invention.

In addition to the criteria of viability, attenuation andimmunogenicity, the properties of the derivative which are selected mustalso be as stable as possible so that the desired attributes aremaintained. Genetic instability of the ts phenotype followingreplication in vivo has been the rule for ts viruses (Murphy et al.,Infect. and Immun. 37:235-242 (1982)). Ideally, then, the virus which isuseful in the vaccines of the present invention must maintain itsviability, its property of attenuation, its ability to replicate in theimmunized host (albeit at lower levels), and its ability to effectivelyelicit the production of an immune response in the vaccinee that issufficient to confer protection against serious disease caused bysubsequent infection by wild-type virus. Clearly, the heretofore knownand reported RS virus mutants do not meet all of these criteria. Indeed,contrary to expectations based on the results reported for knownattenuated RS viruses, some of the viruses of the invention, which havea minimun of two to three distinct mutations, are not only viable andmore attenuated then previous mutants, but are more stable geneticallyin vivo than those previously studied mutants, retaining the ability tostimulate a protective immune response and in some instances to expandthe protection afforded by multiple modifications, e.g., induceprotection against different viral strains or subgroups, or protectionby a different immunologic basis, e.g., secretory versus serumimmunoglobulin, cellular immunity, and the like.

Propagation of the attenuated virus of the invention may be in a numberof cell lines which allow for RS virus growth RS virus grows in avariety of human and animal cells. Preferred cell lines for propagationof attenuated RS virus for vaccine use include DBS-FRhL-2, MRC-5, andVero cells. Highest virus yields are usually achieved with heteroploidcell lines such as Vero cells. Cells are typically inoculated with virusat a multiplicity of infection ranging from about 0.001 to 1.0 or more,and are cultivated under conditions permissive for replication of thevirus, e.g., at about 30-37° C. and for about 3-5 days, or as long asnecessary for virus to reach an adequate titer. Virus is removed fromcell culture and separated from cellular components, typically by wellknown clarification procedures, e.g., centrifugation, and may be furtherpurified as desired using procedures well known to those skilled in theart.

RS virus which has been attenuated as described herein can be tested inin vitro and in vivo models to confirm adequate attenuation, geneticstability, and immunegencity for vaccine use. In in vitro assays, themodified virus is tested for temperature sensitivity of virusreplication, i.e. ts phenotype, and for the small plaque phenotype.Modified viruses are further tested in animal models of RS infection. Avariety of animal models have been described and are summarized inMeignier et al., eds., Animal Models of Respiratory Syncytial VirusInfection, Merieux Foundation Publication, (1991), which is incorporatedherein by reference. A cotton rat model of RS infection is described inU.S. Pat. No. 4,800,078 and Prince et al., Virus Res. 3:193-206 (1985),which are incorporated herein by reference, and is believed to bepredictive of attenuation and efficacy in humans. A primate model of RSinfection using the chimpanzee is predictive of attenuation and efficacyin humans, and is described in detail in Richardson et al., J. Med.Virol. 3:91-100 (1978); Wright et al., Infect. Immun., 37:397-400(1982); Crowe et al., Vaccine 11:1395-1404 (1993), which areincorporated herein by reference.

The interrelatedness of data derived from rodents and chimpanzeesrelating to the level of attenuation of RSV candidates can bedemonstrated by reference to FIG. 1, which is a graph correlating thereplication of a spectrum of respiratory syncytial subgroup A viruses inthe lungs of mice with their replication in chimpanzees. The replicationis substantially identical, allowing the mouse to serve as a model inwhich to initially characterize the level of attenuation of the vaccineRSV candidate. The mouse and cotton rat model are especially useful inthose instances in which candidate RS viruses display inadequate growthin chimpanzees. The RSV subgroup B viruses are an example of the RSviruses which grow poorly in chimpanzees.

Moreover, the therapeutic effect of RSV neutralizing antibodies ininfected cotton rats has been shown to be highly relevant to subsequentexperience with immunotherapy of monkeys and humans infected with RSV.Indeed, the cotton rat appears to be a reliable experimental surrogatefor the response of infected monkeys, chimpanzees and humans toimmunotherapy with RSV neutralizing antibodies. For example, the amountof RSV neutralizing antibodies associated with a therapeutic effect incotton rats as measured by the level of such antibodies in the serum oftreated animals (i.e., serum RSV neutralization titer of 1:302 to 1:518)is in the same range as that demonstrated for monkeys (i.e., titer of1:539) or human infants or small children (i.e., 1:877). A therapeuticeffect in cotton rats was manifest by a one hundred fold or greaterreduction in virus titer in the lung (Prince et al., J. Virol.61:1851-1854) while in monkeys a therapeutic effect was observed to be a50-fold reduction in pulmonary virus titer. (Hemming et al., J. Infect.Dis. 152:1083-1087 (1985)). Finally, a therapeutic effect in infants andyoung children who were hospitalized for serious RSV bronchiolitis orpneumonia was manifest by a significant increase in oxygenation in thetreated group and a significant decrease in amount of RSV recoverablefrom the upper respiratory tract of treated patients. (Hemming et al.,Antimicrob. Agents Chemother. 31:1882-1886 (1987)). Therefore, based onthese studies, it would appear that the cotton rat constitutes arelevant model for predicting the success of an RSV vaccine in infantsand small children. Other rodents, including mice, should also besimilarly useful because these animals are permissive for RSVreplication and have a core temperature more like that of humans (Wightet al., J. Infect. Dis. 122:501-512 (1970) and Anderson et al., J. Gen.Virol. 71:(1990)).

For vaccine use, the attenuated virus of the invention can be useddirectly in vaccine formulations, or lyophilized, as desired, usinglyophilization protocols well known to the artisan. Lyophilized viruswill typically be maintained at about 4° C. When ready for use thelyophihized virus is reconstituted in a stabilizing solution, e.g.,saline or comprising SPG, Mg⁺⁺ and HEPES, with or without adjuvant, asfurther described below.

Thus RS virus vaccines of the invention contain as an active ingredientan immunogenically effective amount of an attenuated RS virus asdescribed herein. The attenuated virus may be introduced into a host,particularly humans, with a physiologically acceptable carrier and/oradjuvant. Useful carriers are well known in the art, and include, e.g.,water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid andthe like. The resulting aqueous solutions may be packaged for use as is,or lyophilized, the lyophilized preparation being combined with asterile solution prior to administration, as mentioned above. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such aspH adjusting and buffering agents, tonicity adjusting agents, wettingagents and the like, for example, sodium acetate, sodium lactate, sodiumchloride, potassium chloride, calcium chloride, sorbitan monolaurate,triethanolainne oleate, and the like.

Upon inoculation with an attenuated RS virus composition as describedherein, via aerosol, droplet, coarse spray, oral, topical or otherroute, most preferably suitable for intranasal delivery, the immunesystem of the host responds to the vaccine by producing antibodies, bothsecretory and serum, specific for RS virus proteins. As a result of thevaccination the host becomes at least partially or completely immune toRS virus infection, or resistant to developing moderate or severe RSviral infection, particularly of the lower respiratory tract.

The vaccine compositions containing the attenuated RS virus of theinvention are administered to a person susceptible to or otherwise atrisk of RS virus infection to enhance the individual's own immuneresponse capabilities. Such an amount is defined to be a“immunogenically effective dose.” In this use, the precise amounts againdepend on the patient's state of health and weight, the mode ofadministration, the nature of the formulation, etc., but generally rangefrom about 10³ to about 10⁶ plaque forming units (PFU) or more of virusper patient, more commonly from about 10⁴ to 10⁵ PFU virus per patient.In any event, the vaccine formulations should provide a quantity ofattenuated RS virus of the invention sufficient to effectively protectthe patient against serious or life-threatening RS virus infection.

The attenuated RS virus of the invention of one particular RS subgroupor strain can be combined with attenuated viruses of the other subgroupor strains to achieve protection against multiple RS viruses. Typicallythe different modified viruses will be in admixture and administeredsimultaneously, but may also be administered separately. Due to thephenomenon of cross-protection among certain strains of RS virus,immunization with one strain may protect against several differentstrains of the same or different subgroup.

In some instances it may be desirable to combine the attenuated RS virusvaccines of the invention with vaccines which induce protectiveresponses to other agents, particularly other childhood viruses. Forexample, the attenuated virus vaccine of the present invention can beadministered simultaneously (typically separately) or sequentially withparainfluenza virus vaccine, such as described in Clements et al., J.Clin Microbiol. 29:1175-1182 (1991), which is incorporated herein byreference.

Single or multiple administrations of the vaccine compositions of theinvention can be carried out. In neonates and infants, multipleadministration may be required to elicit sufficient levels of immunity.Administration should begin within the first month of life, and continueat intervals throughout childhood, such as at two months, six months,one year and two years, as necessary to maintain sufficient levels ofprotection against native (wild-type) RS virus infection. Similarly,adults who are particularly susceptible to repeated or serious RS virusinfection, such as, for example, health care workers, day care workers,family members of young children, elderly, individuals with compromisedcardiopulmonary function, etc. may require multiple immunizations toestablish and/or maintain protective imune responses. Levels of inducedimmunity can be monitored by measuring amounts of neutralizing secretoryand serum antibodies, and dosages adjusted or vaccinations repeated asnecessary to maintain desired levels of protection.

The following examples are provided by way of illustration, notlimitation.

EXAMPLE I Isolation and Characterization of Mutagenized Derivatives ofCold-passaged RSV

This Example describes the chemical mutagenesis of incompletelyattenuated host range-restricted cpRSV to produce derivative ts and spstrains which are more highly attenuated and thus are preferred for usein RSV vaccine preparations.

A parent stock of cold-passaged RSV (cpRSV) was prepared. FlowLaboratories Lot 3131 virus, the cpRSV parent virus that is incompletelyattenuated in humans, was passaged twice in MRC-5 cells at 25° C.,terminally diluted twice in MRC-5 cells at 25° C., then passaged threetimes in MRC-5 to create a cpRSV suspension for mutagenesis.

The cpRSV was mutagenized by growing the parent stock in MRC-5 cells at32° C. in the presence of 5-fluorouracil in the medium at aconcentration of 4×10⁻⁴M. This concentration was demonstrated to beoptimal in preliminary studies, as evidenced by a 100-fold decrease invirus titer on day 5 of growth in cell culture, compared to mediumwithout 5-fluorouracil. The mutagenized stock was then analyzed byplaque assay on Vero cells that were maintained under an agar overlay,and after an appropriate interval of incubation, plaques were stainedwith neutral red dye. 854 plaques were picked and the progeny of eachplaque were separately amplified by growth on fresh monolayers of Verocells. The contents of each of the tissue cultures inoculated with theprogeny of a single plaque of cpRSV-mutagenized virus were separatelyharvested when cytopathic effects on the Vero cells appeared maximal.Progeny virus that exhibited the temperature-sensitive (ts) orsmall-plaque (sp) phenotype was sought by titering these plaque pools onHEp-2 cells at 32° C. and 38° C. Any virus exhibiting sp W phenotype(plaque size that was reduced by 50% or more compared to parental virusat 32° C.) or a U phenotype (100fold or greater reduction in titer atrestrictive temperature [37° to 40° C.] compared to 32° C.) wasevaluated further. These strains were biologically cloned by serialplaque-purification on Vero cells three times, then amplified on Verocells. The cloned strains were titered at 32°, 37°, 38°, 39° and 40° C.(in an efficiency of plaque formation (EOP) assay) to confirm their spand ts phenotypes. Because titers of some cloned strains were relativelylow even at the permissive temperature (32°), these viruses werepassaged once in HEp-2 cells to create virus suspensions for in vitroanalysis. The phenotypes of the progeny of the mutagenized cpRSV arepresented on Table 1.

TABLE 1 The efficiency of plaque formation of nine derivatives ofcold-passaged RSV (cpts or cpsp mutants) in HEp-2 cells at permissiveand restrictive temperatures The titer of virus (log₁₀pfu/ml) Shut-offat the indicated temperature (° C.) temperature Small-plaques Virus 3237 38 39 40 (° C.)¹ at 32C A2 wild-type 4.5 4.4 4.5 3.8 3.8 >40  nocp-RSV 6.0 5.8 5.8 6.2 5.4 >40  no ts-1 5.7 4.5 2.7 2.4 1.7* 38 nocpsp-143 4.2* 4.1* 3.8* 3.9* 3.8* >40  yes cpts-368 6.7 6.3 6.1* 5.8**2.0** 40 no cpts-274 7.3 7.1 6.6 5.8* 1.0** 40 no cpts-347 6.2 6.1 5.7*5.5** <0.7 40 no cpts-142 5.7 5.1 4.5* 3.7** <0.7 39 no cpts-299 6.2 5.55.1* 2.0** <0.7 39 no cpts-475 5.4 4.8* 4.2** <0.7 <0.7 39 no cpts-5305.5 4.8* 4.5* <0.7 <0.7 39 no cpts-248 6.3 5.3** <0.7 <0.7 <0.7 38 no¹Shut-off temperature is defined as the lowest restrictive temperatureat which a 100-fold or greater reduction of plaque titer is observed(bold figures in table). *Small-plaque phenotype (<50% wild-type plaquesize) **Pinpoint-plaque phenotype (<10% wild-type plaque size)

One of the mutant progeny had the small plaque phenotype, RSV cpsp-143(sp refers to the small plaque (sp) phenotype), and the remaining mutantprogeny had the ts phenotype. The RSV cpts mutants exhibit a variationin ability to produce plaques in monolayer cultures in vitro over thetemperature range 37° C. to 40° C., with cpts 368 retaining the abilityto produce plaques at 40° C., whereas the most temperature-sensitive(ts) virus, cpts 248, failed to produce plaques at 38° C. Thus, severalof the mutagenized cpRSV progeny exhibit a marked difference from theircpRSV parent vinus with respect to temperature-sensitivity of plaqueformation.

Replication and Genetic Stability Studies In Mice

The level of replication of the cpRSV derived mutants in the upper andlower respiratory tracts of BALB/c mice was studied next (Table 2). Itwas found that cpts 530 and cpts 248, two of the mutants exhibiting thegreatest temperature sensitivity (see Table 1), were about 7- to 12-foldrestricted in replication in the nasal turbinates of the mice (Table 2).However, none of the viruses was restricted in replication in the lungscompared to the cpRSV parent virus. This greater restriction ofreplication in the nasal turbinates than in the lungs is notcharacteristic of ts mutants, which generally are more restricted inreplication in the warmer lower respiratory tract (Richman and Murphy,Rev. Infect. Dis. 1:413-433 (1979). The virus produced in the lungs andnasal turbinates retained the ts character of the input virus (data notpresented). The present findings suggested that the combination of thets mutations on the background of the mutations of the cp parent virushas resulted in cpRSV ts progeny with a higher level of stability of thets phenotype after replication in vivo than had been seen withpreviously studied ts mutants.

To further explore the level of genetic stability of the ts phenotype ofthe cpRSV derived mutants, the efficiency of plaque formation of viruspresent in the lungs and nasal turbinates of nude mice was studied fortwo mutagenized cpRSV progeny that were among the most ts, namely ts 248and ts 530. Nude mice were selected because they are immunocompromiseddue to congenital absence of functional T-cells, and a virus canreplicate for a much longer period of time in these hosts. This longerperiod of replication favors the emergence of virus mutants with alteredphenotype. The virus present on day 12 (NOTE: in normal mice, virus isno longer detectable at this time) was characterized and found to retainan unaltered ts phenotype (Table 3). As expected, the ts-1 mutantincluded in the test as a positive control exhibited an unstable tsphenotype in vivo. Thus, contrary to previous evaluation of ts mutantviruses in rodents, the results show that a high level of stability ofthe ts phenotype of the cpRSV derived mutants following prolongedreplication in rodents was achieved, which represents a significant andheretofore unattained very desirable property in the viruses of theinvention.

TABLE 2 Replication of cpts - RSV mutants in BALB/c mice¹ Virus titer at32° C. (mean log₁₀pfu/g tissue from the tissue of eight animals ±standard error) Shutoff Day 4 Day 5 Animals temperature of Nasal Nasalinfected with virus (° C.) Turbinates Lungs turbinates Lungs A2wild-type >40  5.0 ± 0.16 5.8 ± 0.20 5.0 ± 0.11 5.8 ± 0.19 cp-RSV >40 4.7 ± 0.07 5.3 ± 0.18 4.8 ± 0.16 5.3 ± 0.21 ts-1 38 4.0 ± 0.19 4.7 ±0.27 3.8 ± 0.33 4.9 ± 0.13 cpsp-143 >40  4.5 ± 0.14 4.1 ± 0.37 4.4 ±0.39 4.6 ± 0.39 cpts-368 40 4.8 ± 0.15 5.1 ± 0.35 4.7 ± 0.08 5.4 ± 0.23cpts-274 40 4.2 ± 0.19 5.0 ± 0.15 4.2 ± 0.11 5.1 ± 0.55 cpts-347 40 4.4± 0.32 4.9 ± 0.40 4.5 ± 0.33 5.2 ± 0.35 cpts-142 39 4.1 ± 0.34 5.0 ±0.19 4.3 ± 0.24 5.8 ± 0.40 cpts-299 39 3.9 ± 0.11 5.2 ± 0.15 3.9 ± 0.325.0 ± 0.29 cpts-475 39 4.0 ± 0.18 5.3 ± 0.25 4.1 ± 0.23 4.9 ± 0.42cpts-530 39 3.9 ± 0.18 5.3 ± 0.15 3.9 ± 0.14 5.3 ± 0.19 cpts-248 38 3.9± 0.33 5.1 ± 0.29 4.2 ± 0.13 5.5 ± 0.35 ¹Mice were administered 10^(6.3)p.f.u. intranasally in a 0.1 ml inoculum on day 0, then sacrificed onday 4 or 5.

TABLE 3 The genetic stability of RSV cpts-248 and cpts-530 followingprolonged replication in nude mice Efficiency of plaque formation atindicated temperature of virus present in nasal turbinates (n.t.) orlungs of nude mice sacrificed 12 days after virus administration¹ 32° C.37° C. Mean titer % animals Mean titer Tissue (log₁₀pfu with virus(log₁₀pfu Animals harvest or % animals per gram % animals with alteredper gram infected input virus Number of with virus tissue or ml withvirus ts tissue or ml with tested animals detectable inoculum)detectable phenotype inoculum) cpts-248 n.t. 19 100 3.8 ± 0.34 0 0 <2.0″ lung ″  90 2.0 ± 0.29 0 0 <1.7 cpts-530 n.t. 20 100 3.0 ± 0.26 0 0<2.0 ″ lungs ″ 100 2.4 ± 0.29 0 0 <1.7 ts-1 n.t. 19 100 3.7 ± 0.23 74 74  2.7 ± 0.57 ″ lungs ″ 100 2.5 ± 0.30 74  74  1.8 ± 0.21 Efficiencycpts-248 — — 4.9 — <0.7 of plaque cpts-530 — — 5.5 —  3.7* formationts-1 — — 6.1 —  3.3 of input viruses Efficiency of plaque formation atindicated temperature of virus present in nasal turbinates (n.t.) orlungs of nude mice sacrificed 12 days after virus administration¹ 38° C.40° C. % animals Mean titer % animals Mean titer Tissue with virus(log₁₀pfu with virus (log₁₀pfu Animals harvest or % animals with alteredper gram % animals with altered per gram infected input virus with virusts tissue or ml with virus ts tissue or ml with tested detectablephenotype inoculum) detectable phenotype inoculum) cpts-248 0 n.t. 0<2.0 0 0 <2.0 ″ lungs 0 0 <1.7 0 0 <1.7 cpts-530 n.t. 0 0 <2.0 0 0 <2.0″ lungs 0 0 <1.7 0 0 <1.7 ts-1 n.t. 63  63  2.4 ± 0.36 10  10  2.0 ±0.13 ″ lungs 35  32  1.8 ± 0.15 0 0 <1.7 Efficiency cpts-248 — <0.7 —<0.7 of plaque cpts-530 — <0.7 — <0.7 formation ts-1 —  2.7 — <0.7 ofinput viruses ¹Plaque titers shown represent the mean log₁₀pfu/gramtissue of 19 or 20 samples ± standard error ²Each animal received10^(6.3) p.f.u. intranasally in 0.1 ml inoculum of the indicated viruson day 0. *Small-plaque phenotype only.

In Chimpanzees

The level of attenuation of the cpRSV ts progeny was next evaluated inthe seronegative chimpanzee, a host most closely related to humans.Trials in chimpanzees or owl monkeys are conducted according to thegeneral protocol of Richardson et al., J. Med. Virol. 3:91-100 (1979);Croweet al., Vaccine 11:1395-1404 (1993), which are incorporated hereinby reference. One ml of suspension containing approximately 10⁴plaque-forming units (PFU) of mutagenized, attenuated virus is givenintranasally to each animal. An alternate procedure is to inoculate theRSV into both the upper and lower respiratory tract at a dose of 10⁴ PFUdelivered to each site. Chimpanzees are sampled daily for 10 days, thenevery 3-4 days through day 20. The lower respiratory tract ofchimpanzees can be sampled by tracheal lavage according to the protocolof Snyder et al., J. Infec. Dis. 154:370-371 (1986) and Crowe et al.,Vaccine 11:1395-1404(1993). Some animals are challenged 4 to 6 weekslater with the wild-type virus. Animals are evaluated for signs ofrespiratory disease each day that nasopharyngeal specimens are taken.Rhinorrhea is scored from 0 to 4+, with 2+ or greater being consideredas evidence of significant upper respiratory disease.

Virus is isolated from nasal and throat swab specimens and tracheallavage fluids by inoculation into RSV-sensitive HEp-2 cells as describedabove. Quantities of virus can also be determined directly by the plaquetechnique using HEp-2 cells as described in Schnitzer et al., J. Virol.17:431-438 (1976), which is incorporated herein by reference. Specimensof serum are collected before administration of virus and at 3 to 4weeks post-inoculation for determination of RSV neutralizing antibodiesas described in Mills et al., J. Immunol. 107:123-130 (1970), which isincorporated herein by reference.

The most ts and attenuated of the cpRSV progeny (cpts 248) was studiedand compared to wild-type RSV and the cpRSV parent virus (Table 4).Replication of the cpRSV parent virus was slightly reduced in thenasopharynx compared to wild-type, there was a reduction in the amountof rhinorrhea compared to wild-type virus, and there was an approximate600-fold reduction in virus replication in the lower respiratory tractcompared to wild-type. Clearly, the cp virus was significantlyrestricted in replication in the lower respiratory tract of chimpanzees,a very desirable property not previously identified from priorevaluations of cpRSV in animals or humans. More significantly, the cpts248 virus was 10-fold restricted in replication in the nasopharynxcompared to wild-type, and this restriction was associated with a markedreduction of rhinorrhea These findings indicated that the cpRSV derivedmutant possesses two highly desirable properties for a live RSV vaccine,namely, evidence of attenuation in both the upper and the lowerrespiratory tracts of highly susceptible seronegative chimpanzees. Thelevel of genetic stability of the virus present in the respiratory tractof chimpanzees immunized with cpts-248 was evaluated next (Table 5). Thevirus present in the respiratory tract secretions retained the tsphenotype, and this was seen even with the virus from chimpanzee No. 3on day 8 that was reduced 100-fold in titer at 40° C. and exhibited thesmall plaque phenotype at 40° C., indicating that its replication wasstill temperature-sensitive. This represents the most genetically stablets mutant identified prior to the time of this test. The increasedstability of the ts phenotype of the cpts 248 and cpts 530 virusesreflects an effect of the cp mutations on the genetic stability of themutations that contribute to the ts phenotype in vivo. Thus, the tsmutations in the context of the mutations already present in the cp3131parent virus appear to be more stable than would be expected in theirabsence. This important property has not been previously observed orreported. Infection of chimpanzees with the cpts 248 induced a hightiter of serum neutralizing antibodies, as well as antibodies to the Fand G glycoproteins (Table 6). Significantly, immunization with cpts 248protected the animals from wild-type RSV challenge (Table 7), indicatingthat this mutant functions as an effective vaccine virus in a host thatis closely related to humans.

These above-presented findings indicate that the cpts 248 virus has manyproperties desirable for a live RSV vaccine, including: 1) attenuationfor the upper and lower respiratory tract; 2) increased geneticstability after replication in vivo, even after prolonged replication inimmunosuppressed animals; 3) satisfactory immunogenicity; and 4)significant protective efficacy against challenge with wild-type RSV.The cpts 530 virus shares with cpts 248 similar temperature sensitivityof plaque formation, a similar degree of restriction of replication inthe nasal turbinates of mice, and a high level of genetic stability inimmunodeficient nude mice, whereby it also represents an RS virusvaccine strain.

TABLE 4 Replication of cpts-RSV 248, cp-RSV, or wild-type RSV A2 in theupper and lower respiratory tract of seronegative chimpanzees Virusrecovery Animal infected Nasopharynx Trachea Rhinorrhea with indicatedRoute of Chimpanzee Duration^(b) Peak titer Duration^(b) Peak titerscore virus Inoculation number (days) (log₁₀pfu/ml) (days) (log₁₀pfu/ml)Mean^(c) Peak cpts-248 IN + IT 1 10 4.6  8^(d) 5.4 0.2 1 IN + IT 2 104.5 6 2.2 0.1 1 IN + IT 3  9 4.7 10  2.1 0.1 1 IN + IT 4  9 4.2  8^(d)2.2 0.1 1 mean 9.5 mean 4.5 mean 8.0 mean 3.0 mean 0.1 cp-RSV IN 5 205.3  8^(d) 2.9 1.0 3 IN 6 16 5.8  6^(d) 3.0 1.8 3 IN + IT 7 13 4.3 6^(d) 3.0 0.6 1 IN + IT 8 16 5.0 10^(d) 2.8 0.5 1 mean 16 mean 5.1 mean7.5 mean 2.9 mean 1.0 A2 wild-type IN 9  9 5.1 13  5.4 1.0 1 IN 10   96.0 8 6.0 1.7 4 IN + IT 11  13 5.3 8 5.9 2.1 3 IN + IT 12   9 5.4 8 5.61.0 3 mean 10 mean 5.5 mean 9.3 mean 5.7 mean 1.4 ^(a)IN = Intranasaladministration only, at a dose of 10⁴ p.f.u. in a 1.0 ml inoculum; IN +IT = Both intranasal and intratracheal administration, 10⁴ p.f.u. in a1.0 ml inoculum at each site. ^(b)Indicates last day post-infection onwhich virus was recovered. ^(c)Mean rhinorrhea score represents the sumof daily scores for a period of eight days surrounding the peak day ofvirus shedding, divided by eight. Four is the highest score; zero is thelowest score ^(d)Virus isolated only on day indicated.

TABLE 5 Genetic stability of virus present in original nasopharyngeal(NP) swabs or tracheal lavage (TL) specimens obtained from animalsexperimentally infected with cpts-RSV 248 Titer of RSV at indicatedVirus obtained temperature (log₁₀pfu/ml) Chimpanzee NP swab or on post-Titer at Titer at Titer at number TL specimen infection day 32° C. 39°C. 40° C.  1^(a) NP 3 3.2 <0.7 NT ″ 4 2.7 <0.7 NT ″ 5 4.2 <0.7 NT ″ 63.8 <0.7 NT ″ 7 4.6 <0.7 NT ″ 8 4.5 <0.7 NT ″ 9 2.6 <0.7 NT ″ 10  2.0<0.7 NT TL 6 5.4 <0.7 NT ″ 8 2.7 <0.7 NT  2^(a) NP 3 3.2 <0.7 NT ″ 4 3.7<0.7 NT ″ 5 4.5 <0.7 NT ″ 6 4.1 <0.7 NT ″ 7 3.3 <0.7 NT ″ 8 4.2 <0.7 NT″ 9 2.8 <0.7 NT ″ 10  1.6 <0.7 NT TL 6 2.2 <0.7 NT 3 NP 3 2.7 <0.7 <0.7″ 4 3.4 <0.7 <0.7 ″ 5 2.9 <0.7 <0.7 ″ 6 3.3 <0.7 <0.7 ″ 7 3.4 0.7^(b)<0.7 ″ 8 4.7 3.5^(b) 2.0^(c) ″ 9 1.9 <0.7 <0.7 TL 6 1.8 <0.7 <0.7 ″ 81.9 1.2^(b) <0.7 ″ 10  2.1 1.3^(b) <0.7 4 NP 3 3.2 <0.7 NT ″ 4 2.7 <0.7NT ″ 5 3.4 <0.7 NT ″ 6 3.3 <0.7 NT ″ 7 4.2 <0.7 NT ″ 8 3.5 <0.7 NT ″ 92.1 <0.7 NT TL 8 2.2 <0.7 NT NT = Not tested ^(a)Isolates (once-passagedvirus suspensions with average titer log₁₀pfu/ml of 4.0) were generatedfor samples from these chimpanzees from each original virus-containingnasopharyngeal swab specimen or tracheal lavage specimen and tested forefficiency of plaque formation at 32°, 39° and 40° C. No isolate wasable to form plaques at 39° C. or 40° C. Isolates from chimpanzees 3 and4 were not tested in this manner. ^(b)The percent titer at 39° C. versusthat at 32° C.: NP swab day 7 = 0.2%, NP swab day 8 = 6%, TL day day 8 =20%, TL day 10 = 16%. All plaques were of small-plaque phenotype only;no wild-type size plaques seen. ^(c)The percent titer at 40° C. versusthat at 32° C. was 0.2%. All plaques were of pinpoint-plaque phenotype;wild-type size plaques were not detected.

TABLE 6 Serum antibody responses of chimpanzees infected with RSVcpts-248, cp-RSV, or RSV A2 wild-type Animals Serum antibody titers(reciprocal mean log₂) immunized Number of Neutralizing ELISA-F ELISA-Gwith chimpanzees Day 0 Day 28 Day 0 Day 28 Day 0 Day 28 cpts-248 4 <3.310.7  7.3 15.3 6.3  9.8 cp-RSV 4 <3.3 11.2 11.3 15.3 9.3 12.3 RSVA2 4<3.3 11.2 8.3 15.3 7.3 10.3 wild-type

TABLE 7 Immunization of chimpanzees with cpts-248 induces resistance toRSV A2 wild-type virus challengge on day 28 Response to challenge with10⁴ p.f.u. wild-type virus administered on day 28 Serum neutral- izingantibody Virus Recovery (reciprocal log₂) Virus used to NasopharynxTrachea Rhinorrhea on day indicated immunize Chimpanzee Peak titer Peaktiter score Day 42 animal number Duration (days) (log₁₀pfu/ml) Duration(days) (log₁₀pfu/ml) Mean^(a) Peak Day 28 or 56 cpts-248 1 5 2.7 0 <0.70 0 10.1 11.0 2 9 1.8 0 <0.7 0 0 10.3 14.5 cp-RSV 5 5 1.0 0 <0.7 0 011.1 13.3 6 8 0.7 0 <0.7 0 0 11.4 12.9 none 9 9 5.1 13 5.4 1.0 1 <3.312.4 10 9 6.0 8 6.0 1.7 4 <3.3 13.2 11 13 5.3 8 5.9 2.1 3 <3.3 11.6 12 95.4 8 5.6 1.0 3 <3.3 11.2 ^(a)Mean rhinorrhea score represents the sumof scores during the eight days of peak virus shedding divided by eight.Four is the highest score. A score of zero indicates no rhinorheadetected on any day of the ten-day observation period.

Further Attenuations

Since RS virus produces more symptoms of lower respiratory tract diseasein human infants than in the 1-2 year old chimpanzees used in theseexperimental studies, and recognizing that mutants which aresatisfactorily attenuated for the chimpanzee may not be so forseronegative infants and children, the cpts 248 and 530 derivatives,which possess the very uncharacteristic ts mutant properties ofrestricted replication and attenuation in the upper respiratory tractand a higher level of genetic stability, were further mutagenized.

Progeny viruses that exhibited a greater degree oftemperature-sensitivity in vitro than cpts 248 or that had the smallplaque phenotype were selected for further study. Mutant derivatives ofthe cpts 248 that possessed one or more additional ts mutations wereproduced by 5-fluorouracil mutagenesis (Table 8). Ts mutants that weremore temperature-sensitive (ts) than the cpts 248 were identified, andsome of these had the small plaque (sp) phenotype. These cpts 248derivatives were administered to mice. Cpts 248/804, 248/955, 248/404,248/26, 248/18, and 248/240 mutants were more restricted in replicationin the upper and lower respiratory tract of the mouse than their cpts248 parental virus (Table 9). Thus, viable mutants of cpts 248 whichwere more attenuated than their cpts 248 were identified, and thesederivatives of cpts 248 exhibited a wide range of replicative efficiencyin mice, with ts 248/26 being the most restricted. The ts phenotype ofthe virus present in nasal turbinates and lungs of the mice was almostidentical to that of the input virus, indicating genetic stability. Ahighly attenuated derivative of cpts 248, the cpts 248/404 virus, was1000-fold more restricted in replication in the nasopharynx compared towild-type. The cpts 248/404 mutant, possessing at least threeattenuating mutations, was also highly restricted in replication in theupper and lower respiratory tracts of four seronegative chimpanzees andinfection did not induce rhinorrhea (Table 10). Again, this virusexhibited a high degree of reduction in replication compared towild-type, being 60,000-fold reduced in the nasopharynx and 100,000-foldin the lungs. Nonetheless, two chimpanzees which were subsequentlychallenged with RSV wild-type virus were highly resistant (Table 11).

Five small-plaque mutants of cpts-248/404 were derived by chemicalmutagenesis in a similar fashion to that described above. Suspensions ofonce-amplified plaque progency were screened for the small-plaque (sp)phenotype by plaque titration at 32° C. on HEp-2 cells, and workingsuspensions of virus were prepared as described above.

Five of the 1785 plaque progeny of the mutagenized cpts-248/404 virusexhibited a stable sp phenotype. The shut-off temperature of each mutantwas 35° C. or less (Table 12), suggesting that each of these spderivatives of the cpts-248/404 virus also had acquired an additional tsmutation. Following intranasal inoculation of Balb/c mice with 10^(6.3)p.f.u. of a sp derivative of the cpst-248/404, virus could not bedetected in the nasal turbinates of mice inoculated with any of these spderivatives. However, virus was detected in low titer in the lungs inone instance. These results indicate >300-fold restriction ofreplication in the nasal turbinates and >10,000-fold restriction inlungs compared with wild-type RSV.

Further ts derivatives of the cpts 530 virus were also generated (Table13). As with the cpts-248 derivatives, the cpts-530 derivatives weremore restricted in replication in mice than the cpst-530 parentalstrain. One mutant, cpts-530/1009, was 30 times more restricted inreplication in the nasal turbinates of mice. This cpts-530 derivative isalso highly restricted in replication in the upper and lower respiratorytract of seronegative chimpanzees (Table 14). In the nasopharynx,cpts-530 was 30-fold restricted in replication, while cpts-530/1009 was100-fold restricted compared to wild-type virus. Both of the cptsmutants were highly restricted (20,000 to 32,000-fold) in the lowerrespiratory tract compared with wild-type virus, even when the mutantswere inoculated directly into the trachea. Also, chimpanzees previouslyinfected with cpts-530/1009, cpts-530 or cp-RSV exhibited significantrestriction of virus replication in the nasopharynx and did not developsignificant rhinorrhea following subsequent combined intranasal andintratracheal challenge with wild-type RSV (Table 15). In addition,chimpanzees previously infected with any of the mutants exhibitedcomplete resistance in the lower respiratory tract to replication ofwild-type challenge virus.

These results were completely unexpected based on experience gainedduring prior studies. For example, the results of an earlier studyindicated that the in vivo properties of RSV ts mutants derived from asingle cycle of 5-fluorouracil mutagenesis could not be predicted apriori. Moreover, although one of the first four ts mutants generated inthis manner exhibited the same shut off temperature for plaque formationas the other mutants, it was overattenuated when tested in susceptiblechimpanzees and susceptible infants and young children [Wright et al.,Infect Immun. 37 (1):397-400 (1982)]. This indicated that theacquisition of the ts phenotype resulting in a 37°-38° C. shut offtemperature for plaque formation did not reliably yield a mutant withthe desired level of attenuation for susceptible chimpanzees, infantsand children. Indeed, the results of studies with heretofore known tsmutants completely fail to provide any basis for concluding thatintroduction of three independent mutations (or sets of mutations) intoRSV by cold-passage followed by two successive cycles of chemicalmutagenesis could yield viable mutants which retain infectivity forchimpanzees (and by extrapolation, young infants) and exhibit thedesired level of attenuation, immunogenicity and protective efficacyrequired of a live virus vaccine to be used for prevention of RSVdisease.

The above-presented results clearly demonstrate that certain tsderivatives of the cpRSV of the invention have a satisfactory level ofinfectivity and exhibit a significant degree of attenuation for mice andchimpanzees. These ts mutant derivatives are attenuated and appearhighly stable genetically after replication in vivo. These mutants alsoinduce significant resistance to RSV infection in chimpanzees. Thus,these derivatives of cpRSV represent virus strains suitable for use in alive RSV vaccine designed to prevent serious human RSV disease.

TABLE 8 The efficiency of plaque formation of ten mutants derived fromRSV cpts- 248 by additional 5FU mutagenesis The titer of virus(log₁₀pfu/ml) Shut-off Small- at the indicated temperature (° C.)temperature plaques Virus 32 35 36 37 38 39 40 (° C.)¹ at 32C A2wild-type 4.5 4.6 4.4 4.5 4.5 3.8 3.8 >40  no cp-RSV 4.7 4.4 4.3 4.3 4.23.7 3.5 >40  no ts-1 5.6 5.4 4.9 4.4 2.7 2.0 <0.7 38 no cpts-248 3.4 3.02.6* 1.7** <0.7 <0.7 <0.7 38 no 248/1228 5.5* 5.3* 5.3** <0.7 <0.7 <0.7<0.7 37 yes 248/1075 5.3* 5.3* 5.1** <0.7 <0.7 <0.7 <0.7 37 yes 248/9654.5 4.2 4.2* <0.7 <0.7 <0.7 <0.7 37 no 248/967 4.4 3.7 3.6* <0.7 <0.7<0.7 <0.7 37 no 248/804 4.9 4.5 4.0* <0.7 <0.7 <0.7 <0.7 37 no 248/9554.8 3.7 2.8* <0.7 <0.7 <0.7 <0.7 36 no 248/404 3.6 2.9* <0.7 <0.7 <0.7<0.7 <0.7 36 no 248/26 3.1 2.9* <0.7 <0.7 <0.7 <0.7 <0.7 36 no 248/184.0* 4.0** <0.7 <0.7 <0.7 <0.7 <0.7 36 yes 248/240 5.8* 5.7** <0.7 <0.7<0.7 <0.7 <0.7 36 yes ¹Shut-off temperature is defined as the lowestrestrictive temperature at which a 100-fold or greater reduction ofplaque titer in HEp-2 cells is observed (bold figures in table).*Small-plaque phenotype (<50% wild-type plaque size) **Pinpoint-plaquephenotype (<10% wild-type plaque size)

TABLE 9 Replication and genetic stability of ten mutants derived fromRSV cpts-248 in Balb/c mice¹ Shutoff Virus titer (mean log₁₀pfu/g tissueof six animals ± standard error) Virus used to temperature of Nasalturbinates Lungs infect animal virus (° C.) 32° C. 36° C. 37° C. 38° C.32° C. 36° C. 37° C. 38° C. A2 wild-type >40  5.1 ± 0.15 5.2 ± 0.23 5.2± 0.14 5.2 ± 0.27 6.1 ± 0.14 5.8 ± 0,23 6.0 ± 0.12 5.9 ± 0.17cp-RSV >40  4.9 ± 0.20 5.1 ± 0.16 4.9 ± 0.24 4.9 ± 0.22 6.0 ± 0.16 5.9 ±0.23 5.6 ± 0.15 5.6 ± 0.13 ts-1 38 3.9 ± 0.25 2.7 ± 0.27 2.4 ± 0.42 2.5± 0.29 4.1 ± 0.21 3.5 ± 0.23 2.6 ± 0.18 2.0 ± 0.23 cpts-248 38 4.0 ±0.16 2.5 ± 0.34 <2.0 <2.0 4.4 ± 0.37 1.8 ± 0.15 <1.7 <1.7 248/1228 374.1 ± 0.15 2.4 ± 0.48 <2.0 <2.0 2.0 ± 0.37 <1.7 <1.7 <1.7 248/1075 374.2 ± 0.18 2.4 ± 0.40 <2.0 <2.0 5.5 ± 0.16 3.5 ± 0.18 <1.7 <1.7 248/96537 3.8 ± 0.23 <2.0 <2.0 <2.0 4.5 ± 0.21 3.4 ± 0.16 <1.7 <1.7 248/967 374.4 ± 0.20 <2.0 <2.0 <2.0 5.4 ± 0.20 3.6 ± 0.19 <1.7 <1.7 248/804 37 2.9± 0.19 <2.0 <2.0 <2.0 3.6 ± 0.19 <1.7 <1.7 <1.7 248/955 36 3.2 ± 0.10<2.0 <2.0 <2.0 3.2 ± 0.22 <1.7 <1.7 <1.7 248/404 36  2.1 ± 0.31² <2.0<2.0 <2.0  4.4 ± 0.12² 1.8 ± 0.20 <1.7 <1.7 248/26 36 <2.0 <2.0 <2.0<2.0 2.3 ± 0.20 <1.7 <1.7 <1.7 248/18 36 2.9 ± 0.99 <2.0 <2.0 <2.0 4.3 ±0.23 1.8 ± 0.15 <1.7 <1.7 248/240 36 2.9 ± 0.82 <2.0 <2.0 <2.0 3.9 ±0.12 <1.7 <1.7 <1.7 ¹Mice were administered 10^(6.3) p.f.u. intranasallyunder light anesthesia on day 0, then sacrificed by CO₂ asphyxiation onday 4. ²In a subsequent study, the level of replication of thecpts-248/404 virus was found to be 2.4 ± 0.24 and 2.6 ± 0.31 in thenasal turbinates and lungs respectively.

TABLE 10 Replication of cpts-RSV 248/404, cpts 248/18, cpts-RSV 248,cp-RSV, or wild-type RSV A2 in the upper and lower respiratory tract ofseronegative chimpanzees Virus recovery Animal infected NasopharynxTrachea Rhinorrhea with indicated Route of Chimpanzee Duration^(b) Peaktiter Duration^(b) Peak titer scores virus Inoculation number (days)(log₁₀pfu/ml) (days) (log₁₀pfu/ml) Mean^(c) Peak cpts-248/404 IN + IT 130 <0.7 0 <0.7 0 0 IN + IT 14 0 <0.7 0 <0.7 0 0 IN + IT 15 8 1.9 0 <0.70.3 2 IN + IT 16 9 2.0 0 <0.7 0.2 1 mean 4.3 mean 1.3 mean 0 mean <0.7mean 0.1 mean 0.8 cpts-248# IN + IT 1 10 4.6  8^(d) 5.4 0.2 1 IN + IT 210 4.5 6 2.2 0.1 1 IN + IT 3 9 4.7 10  2.1 0.1 1 IN + IT 4 9 4.2  8^(d)2.2 0.1 1 mean 9.5 mean 4.5 mean 8.0 mean 3.0 mean 0.1 mean 1.0 cp-RSV#IN 5 20 5.3  8^(d) 2.9 1.0 3 IN 6 16 5.8  6^(d) 3.0 1.8 3 IN + IT 7 134.3  6^(d) 3.0 0.6 1 IN + IT 8 16 5.0 10^(d) 2.8 0.5 1 mean 16 mean 5.1mean 7.5 mean 2.9 mean 1.0 mean 2.0 A2 wild-type# IN 9 9 5.1 13  5.4 1.01 IN 10 9 6.0 8 6.0 1.7 4 IN + IT 11 13 5.3 8 5.9 2.1 3 IN + IT 12 9 5.48 5.6 1.0 3 mean 10 mean 5.5 mean 9.3 mean 5.7 mean 1.4 mean 2.8 ^(a)IN= Intranasal only; IN + IT = Both intranasal and intratrachealadministration. ^(b)Indicates last day post-infection on which virus wasrecovered. ^(c)Mean rhinorrhea score represents the sum of daily scoresfor a period of eight days surrounding the peak day of virus shedding,divided by eight. Four is the highest score; zero is the lowest score.^(d)Virus isolated only on day indicated. # These are the same animalsincluded in Tables 4 and 7.

TABLE 11 Immunization of chimpanzees with cpts-248/404 inducesresistance to RSV A2 wild-type virus challenge on day 28. Serumneutralizing antibody titer Virus Recovery (reciprocal log₂) on Virusused to Nasopharynx Trachea Rhinorrhea on day indicated^(b) immunizeChimpanzee Duration Peak titer Duration Peak titer score Day 49 animalnumber (days) (log₁₀pfu/ml) (days) (log₁₀pfu/ml) Mean^(a) Peak Day 28 or56 cpts-248/404 13 0 <0.7 0 <0.7 0 0 7.9 9.0 14 8 3.4 0 <0.7 0 0 7.012.5 mean 4.0 mean 2.0 mean 0 mean <0.7 mean 0 mean 0 mean 7.5 mean 10.8cp-ts-248# 1 5 2.7 0 <0.7 0 0 11.5 13.0 2 9 1.8 0 <0.7 0 0 12.7 14.5mean 7.0 mean 2.3 mean 0 mean <0.7 mean 0 mean 0 mean 12.1 mean 13.8cp-RSV# 5 5 1.0 0 <0.7 0 0 12.2 11.1 6 8 0.7 0 <0.7 0 0 11.9 9.9 mean6.5 mean 0.9 mean 0 mean <0.7 mean 0 mean 0 mean 12.1 mean 10.5 None # 99 5.1 13 5.4 1.0 1 <3.3 11.0 10 9 6.0 8 6.0 1.7 4 <3.3 9.8 11 13 5.3 85.9 2.1 3 <3.3 9.4 12 9 5.4 8 5.6 1.0 3 <3.3 14.5 mean 10 mean 5.5 mean9.2 mean 5.7 mean 1.4 mean 2.8 mean <3.3 mean 11.2 ^(a)Mean rhinorrheascore represents the sum of scores during the eight days of peak virussheeding divided by eight. Four is the highest score. # These are thesame animals included in Tables 4, 7, and 10. ^(b)Serum nuetralizingtiters in this table, including those from animals previously described,were determined simultaneously in one assay.

TABLE 12 The efficiency of plaque formation and replication in Balb/cmice of five small-plaque derivatives of RSV cpts-248/404. Efficiency ofplaque formation tested in HEp-2 cells at permissive and restrictivetemperatures The titer of virus (log₁₀pfu/ml) Shut-off Small- at theindicated temperature (° C.) temp. plaques Replication in Balb/c mice²Virus 32 35 36 37 38 39 40 (° C.)¹ at 32° C. Nasal turbinates³ Lungs³ A2wild-type 6.0 6.1 6.0 5.8 5.9 5.4 5.4 >40 no 4.5 ± 0.34 5.6 ± 0.13cp-RSV 6.2 6.2 6.0 6.0 5.9 5.6 5.4 >40 no 4.5 ± 0.10 5.3 ± 0.20 cpts-2487.5 7.3 6.2** 5.3** <0.7 <0.7 <0.7  37 no 3.3 ± 0.35 4.8 ± 0.14 248/4045.5 3.6** <0.7 <0.7 <0.7 <0.7 <0.7  36 no 2.4 ± 0.24 2.6 ± 0.31248/404/774 2.9* <0.7 <0.7 <0.7 <0.7 <0.7 <0.7 ≦35 yes <2.0 1.8 ± 0.24248/404/832 5.5** <0.7 <0.7 <0.7 <0.7 <0.7 <0.7 ≦35 yes <2.0 <1.7248/404/886 5.0** <0.7 <0.7 <0.7 <0.7 <0.7 <0.7 ≦35 yes <2.0 <1.7248/404/893 5.4** <0.7 <0.7 <0.7 <0.7 <0.7 <0.7 ≦35 yes <2.0 <1.7248/404/1030 4.4* 2.2** <0.7 <0.7 <0.7 <0.7 <0.7  35 yes <2.0 <1.7¹Shut-off temperature is defined as the lowest restrictive temperatureat which a 100-fold or greater reduction of plaque titer is observedbold figures in table). ²Mice were administered 10^(6.3) p.f.u.intranasally under light anesthesia on day 0, then sacrificed by CO₂asphyxiation on day 4 when tissues were harvested for virus titer. ³Meanlog₁₀pfu/g tissue of six animals ± standard error. *Small-plaquephenotype (<50% wild-type plaque size). **Pinpoint-plaque phenotype(<10% wild-type plaque size).

TABLE 13 The efficiency of plaque formation and level of replication inmice of 14 mutants derived from RSV cpts-530, compared with controlsReplication in mice² (mean In vitro efficiency of plaque formationlog₁₀pfu/g tissue The titer of virus (log₁₀pfu/ml) at the indicatedShut-off of six animals ± SE temperature (° C.) Temp. Nasal RSV 32 34 3536 37 38 39 40 (° C.)¹ turbinates Lungs A2 6.3 6.3 6.1 6.2 6.3 6.3 6.15.6 >40  5.0 ± 0.14 5.8 ± 0.05 cp-RSV 6.5 6.2 6.2 6.2 6.1 6.0 6.15.6 >40  n.d. n.d. cpts-248 6.3 6.3 6.3 6.3 3.7** <0.7 <0.7 <0.7 37/384.1 ± 0.08 5.1 ± 0.13 248/404 6.3 5.7* 4.3** <0.7 <0.7 <0.7 <0.7 <0.735/36 2.1 ± 0.19 3.6 ± 0.10 cpts-530 6.2 6.3 6.2 6.1 6.2* 5.5** <0.7<0.7 39 3.4 ± 0.09 4.3 ± 0.14 530/346 5.9 5.9 5.7 4.7 3.5 <0.7 <0.7 <0.737 3.3 ± 0.11 4.7 ± 0.09 530/977 5.0 4.4 3.6 3.4 2.8* <0.7 <0.7 <0.7 373.4 ± 0.11 2.7 ± 0.04 530/9 6.0 5.6 5.0 3.5* 3.5** <0.7 <0.7 <0.7 36 2.1± 0.06 3.5 ± 0.08 530/1009 4.8 4.0 3.7* 2.0** 1.5** <0.7 <0.7 <0.7 362.2 ± 0.15 3.5 ± 0.13 530/667 5.5 4.9 4.5* 2.0** 0.7 <0.7 <0.7 <0.7 362.4 ± 0.12 2.9 ± 0.15 530/1178 5.7 4.0 5.5 3.7** 2.0** <0.7 <0.7 <0.7 363.3 ± 0.06  42 ± 0.11 530/464 6.0 5.0* 4.7* 1.8** <0.7 <0.7 <0.7 <0.7 36<2.0 2.6 ± 0.10 530/403 5.7 5.1 4.3 2.9 <0.7 <0.7 <0.7 <0.7 36 <2.0 <1.7530/1074 5.1 4.6 4.1* <0.7 <0.7 <0.7 <0.7 <0.7 36 3.0 ± 0.13 3.8 ± 0.13530/963 5.3 5.0 4.2* 0.7 <0.7 <0.7 <0.7 <0.7 36 2.0 ± 0.05 <1.7 530/6535.4 5.1 4.5 <0.7 <0.7 <0.7 <0.7 <0.7 36 2.2 ± 0.10 3.1 ± 0.16 530/10035.6 4.1 2.5 2.1** <0.7 <0.7 <0.7 <0.7 35 <2.0 <1.7 530/1030 4.3 3.7*1.7** <0.7 <0.7 <0.7 <0.7 <0.7 35 <2.0 1.8 ± 0.13 530/188 5.0* 1.0* 1.0<0.7 <0.7 <0.7 <0.7 <0.7 ≦34  <2.0 <1.7 n.d. = not done *Small-plaquephenotype (<50% wild-type plaque size) **Pinpoint-plaque phenotype (<10%wild-type plaque size) ¹Shut-off temperature is defined as the lowestrestrictive temperature at which a 100-fold or greater reduction ofplaque titer is observed (bold figures in table). ²Mice wereadministered 10^(6.3) p.f.u. intranasally under light anesthesia on day0, then sacrificed by CO₂ asphyxiation on day 4.

TABLE 14 Replication of cpts-530/1009, cpts-RSV 530, cp-RSV, orwild-type RSV A2 in the upper and lower respiratory tract ofseronegative chimpanzees induces serum neutralizing antibodies. Virusreplication Animal infected Nasopharynx Trachea Rhinorrhea Day 28reciprocal with 10⁴ pfu of Route of Chimpanzee Duration^(b) Peak titerDuration^(b) Peak titer scores serum neutralizing indicated virusInoculation number (days) (log₁₀pfu/ml) (days) (log₁₀pfu/ml) Mean^(c)Peak antibody titer^(g) cpts-530/1009 IN + IT 1 9 3.1 0 <1.0 0.5 2 1,097IN + IT 2 10  4.0 10^(e) 1.8 0.5 2   416 IN + IT 3 9 4.0 0 <1.0 0.8 21,552 IN + IT 4 9 3.3 0 <1.0 0.4 1 1,176 mean 9.3 mean 3.6 mean 2.5 mean1.2 mean 0.5 mean 1.3 mean 1,060 cpts-530 IN + IT 5 9 3.5  4^(e) 2.6 0.31 10,085  IN + IT 6 9 5.2 0 <1.0 1.1 3 3,566 IN + IT 7 8 3.3 0 <1.0 0.62   588 IN + IT 8 8 4.4 0 <1.0 0.5 2 1,911 mean 8.5 mean 4.1 mean 1.0mean 1.4 mean 0.6 mean 2.0 mean 4,038 cp-RSV IN  9^(d) 20  5.3  8^(e)2.9 1.0 3   416 IN 10^(d) 16  5.8  6^(e) 3.0 1.8 3 2,048 IN + IT 11^(d)13  4.3  6^(e) 3.0 0.6 1   776 IN + IT 12^(d) 16  5.0 10^(e) 2.8 0.5 1  891 mean 16 mean 5.1 mean 7.5 mean 2.9 mean 1.0 mean 2.0 mean 1,033 A2wild-type IN 13^(f) 9 5.1 13  5.4 1.0 1 1,351 IN 14^(f) 9 6.0 8 6.0 1.74   676 IN + IT 15^(d) 13  5.3 8 5.9 2.1 3 1,261 IN + IT 16^(d) 9 5.4 85.6 1.0 3 20,171  mean 10 mean 5.5 mean 9.3 mean 5.7 mean 1.4 mean 2.8mean 5,865 ^(a)IN = Intranasal only; IN + IT = Both intranasal andintratracheal administration. ^(b)Indicates last day post-infection onwhich virus was recovered. ^(c)Mean rhinorrhea score represents the sumof daily scores for a period of eight days surrounding the peak day ofvirus shedding, divided by eight. Four is the highest score; zero is thelowest score. ^(d)Animals from Crowe, et al., Vaccine 12:691-699 (1994).^(e)Virus isolated only on day indicated. ^(f)Animals from Collins, etal. Vaccine 8:164-168 (1990). ^(g)*Determined by complement-enchanced60% plaque reduction of RSV A2 in HEp-2 cell monolayer cultures. Alltiters were determined simultaneously in a single assay. The reciprocaltiter of each animal on day 0 was <10.

TABLE 15 Immunization of chimpanzees with cpts-530/1009 or cpts-530induces resistance to wild-type RSV A2 virus challenge on day 28. Virusreplication Serum neutralizing antibody Nasopharynx Tracheal lavageRhinorrhea (reciprocal log₂ ) on day Virus used for Chimpanzee DurationPeak titer Duration Peak titer scores indicated^(d) immunization number(days) (log₁₀pfu/ml) (days) (log₁₀pfu/ml) Mean^(a) Peak Day 28 Day 49 or56 cpts-530/1009 3 7 2.1 0 <0.7 0 0 1,552 3,823 4 0 <0.7 0 <0.7 0 01,176 1,911 cpts-530 5 0 <0.7 0 <0.7 0 0 10,085 6,654 6 0 <0.7 0 <0.70.3 2 3,566 1,911 cp-RSV 11^(b) 5 1.0 0 <0.7 0 0 776 2,048 12^(b) 8 0.70 <0.7 0 0 891 1,783 none 13^(b) 9 5.1 13  5.4 1.0 1 <10 1,351 14^(b) 96.0 8 6.0 1.7 4 <10   676 15^(c) 13  5.3 8 5.9 2.1 3 <10 1,261 16^(c) 95.4 8 5.6 1.0 3 <10 20,171  ^(a)Mean rhinorrhea scores represent the sumof scores during the eight days of peak virus shedding divided by eight.Four is the highest score. ^(b)Animals from Crowe et al. Vaccine12:691-699 (1994). ^(c)Animals from Collins et al. Vaccine 8:164-168(1990). ^(d)Serum neutralizing titers in this table, including thosefrom animals previously described, were determined simultaneously in oneassay.

Effect of Passively-Acquired Serum RSV Antibodies on cpts Mutants inChimpanzees

In order to examine the effect of passively-acquired serum RSVantibodies on attenuation, immunogenicity and protective efficacy ofvarious cpts mutants of the invention in chimpanzees, the in vivoreplication of cpts-248, cpts-248/404, and cpts-530/1009, was evaluatedin seronegative chimpanzees which were infused with RSV immune globulintwo days prior to immunization (Table 16). Antibody was passivelytransferred in order to simulate the conditions which obtain in younginfants who possess maternally-derived RSV antibodies. In this way, itwas possible to assess the immunogenicity of each indicated mutant inthe presence of passive RSV antibodies to determine whether thereplication of highly attenuated viruses might be so reduced in infantswith a moderate to high titer of passive antibodies as to preclude theinduction of a protective immune response. It would also be possible todefine the nature of the antibody response to immunization in thepresence of passively acquired antibodies, and to define the extent andfunctional activity of the antibody response to virus challenge. Thelevel of virus replication in the nasopharynx and the associatedclinical score for the attenuated mutants was either not altered or onlymoderately altered by the presence of serum RSV antibodies when theinfection of those animals was compared to that of non-infusedseronegative chimpanzees. In contrast, the presence ofpassively-acquired antibodies effectively prevented virus replication ofcpts-248 in the lower respiratory tract. Because the other two mutantswere already highly restricted in the lungs, the similar effect ofpassive antibodies could not be evaluated against those mutants.

Infusion of human RSV immune globulin yielded moderately high serumlevels of RSV F antibodies (titer 1:640 to 1:1600), and neutralizingantibodies (titer 1:199 to 1:252), but not appreciable amounts of serumRSV G antibody detectable above background (Table 17). Chimpanzees whowere infused with human RSV antibodies prior to immunization withcpts-248/404, cpts-530/1009, or cpts-248 developed only one-tenth thequantity of RSV F antibodies and about one-half the titer ofneutralizing antibodies by day 42 post-immunization, compared tonon-infused immunized animals tested 28 days post-immunization. Becausethe infused human IgG contained substantial amounts of RSV F and RSVneutralizing antibodies, the residual antibodies from the infusionpresent in the 42-day serum samples could not be distinguished fromantibodies produced de novo in response to immunization. Given thenormal half-life of human serum IgG antibodies in chimpanzees (Prince etal., Proc. Natl. Acad. Sci. USA 85:6944-6948), the observed levels of Fand neutralizing antibodies on day 42 following immunization with cptsare higher than would be predicted for a residuum of the infusion. Inaddition, the RSV G antibody response following immunization of theinfused animals confirms that these chimpanzees mounted an immuneresponse ts immuization.

Four to six weeks following immunization the chimpanzees were challengedwith wild-type RSV. Each of the animals exhibited complete resistance intheir lower respiratory tract, whether or not human IgG was infused twodays before immunization (Table 18). Non-infused animals developed amodest neutralizing antibody response to challenge or none at all (Table17). In contrast, the infused chimpanzees uniformly developed anunusually high titer of RSV neutralizing antibodies in response towild-type virus challenge despite the fact that virus replication hadbeen severely restricted Crables 17 and 18). Moreover, followingimmunization in the presence of antibodies the most attenuated virus,cpts-248/404, which exhibited the lowest level of virus replicationduring immunization, had the highest post-challenge neutralizingantibody titers (Table 17). In contrast, the least attenuated virus,cpts-248, had the lowest post-challenge neutralizing antibody titer ofthe three groups of infused animals. In addition to an increase in thequantity of the antibodies induced by immunization in the presence ofantibodies, the quality of the antibodies, as measured by theneutralizing to ELISA F antibody titer ratio, was significantly greaterthan that induced by immunization in seronegative animals (Table 17).The neutralizing/ELISA F ratio of the antibodies produced in theinfused/immunized animals post-challenge was about 10- to 20fold higherthan in the non-infused animals and was consistent in all groups,regardless of mutant used to immunize (Table 17).

The presence of passively-acquired antibodies at the time ofimmunization with a live virus vaccine might alter the immune responseto vaccine in three distinct ways. First, a significant decrease in thelevel of replication of vaccine virus might occur that results indecreased immunogenicity. It is possible that the passively-transferredRSV antibodies could restrict the replication of the vaccine viruses,especially the most defective mutants, and greatly decrease theirimmunogenicity. The results presented herein indicate that replicationof the least attenuated mutant (cpts-248) in the lower respiratory tractwas indeed abrogated by the presence of passively-acquired serum IgG RSVantibodies, whereas replication in the upper respiratory tract did notappear to be significantly affected The replication of the leastattenuated mutant tested, cpts-248, was ≧200-fold more (i.e. completely)restricted in the lower respiratory tract in the presence of antibodies.The level of replication of the more attenuated mutants, cpts-530/1009and cpts-248/404, in the lower respiratory tract was highly restrictedeven in the seronegative animals. Therefore, a significant effect ofpassive antibodies on virus replication could not be detectedImmunization with each of the three attenuated mutants induced a highdegree of protection against wild-type challenge in both the upper andlower respiratory tracts, whether or not passively-acquired RSVantibodies were present at the time of immunization. Thus, the level ofreplication of the vaccine viruses in the upper respiratory tract ofpassively-immunized chimpanzees was sufficient to induce a high level ofresistance to wild-type virus challenge which was comparable to thatinduced in non-infused animals.

Second, passive antibodies can alter the immune response to infection bycausing a decrease in the amount and functional activity of antibodiesthat are induced. For this reason the magnitude and the character of theantibody response to live virus immunization in the presence of passiveantibodies was analyzed. Postimmunization serum ELISA IgG F antibodytiters of immunized, infused animals were 10-fold lower than thepostimmunization F titers of non-infused seronegative animals. The serumRSV neutralizing antibody response was also slightly decreased in thoseanimals, on average being 2-fold lower than in non-infused animals.Because some of the ELISA F and neutralizing antibodies detectedpostimmunization represent residual antibodies from the infusion, theactual decrease of the neutralizing and F antibody response caused bypreexisting antibodies is probably even more significant than isapparent. Moreover, the human immune globulin preparation used containeda low level of antibodies to the G glycoprotein of RSV (Table 17). Thispermitted an examination of the IgG RSV G glycoprotein antibody responseof the chimpanzees to infection with the candidate vaccine viruses. TheG antibody responses demonstrated at least a 4-fold or greater increase,indicating that each of the passively-immunized animals was infected byvaccine virus, including chimpanzees immunized with cpts-248/404 whichdid not shed virus. The magnitude of the G antibody response toimmunization did not appear to be adversely influenced by the passivelytransferred antibodies.

Thirdly, the antibody response to RSV wild-type virus challenge ofanimals immunized in the presence of passively-acquired antibodies couldbe altered Chimpanzees immunized in the absence of infused antibodiesexhibited significant resistance to subsequent RSV challenge. Inaddition, these animals failed to develop an appreciable antibodyresponse to challenge virus. Although each of the 6 infused, immunizedanimals also exhibited significant resistance to RSV, a greatly enhancedantibody response to challenge was observed. Post-challenge F or Gantibody levels in the treated animals immunized with cpts-530/1009 orcpts-248/404 were increased at least 10fold, while the neutralizingantibody response represented as much as an 800-fold increase. Theseresults suggest that repeated immunization of infants possessingmaternal antibodies with live attenuated mutants beginning very early inlife might stimulate effective resistance and an associated enhancedsecondary antibody response of high quality. The mechanism responsiblefor an enhanced immune response to second infection in the absence ofappreciable replication of the challenge virus is not understood. Thepresence of serum antibodies at the time of immunization, while allowinga modest antibody response to immunization in infused animals, favorsthe development of a B cell repertoire that elaborates antibodies ofhighly functional activity following subsequent RSV challenge.

The results reported herein are highly significant in that for the firsttime live attenuated RSV virus vaccine has been shown to be efficaciousin an animal model which mimics the target population for an RSVvaccine, i.e. the four to six week old infant having passively acquiredRSV neutralizing antibodies as a result of transplacental transfer fromthe mother. The importance of this finding is clear from the fact that,as discussed, supra the high expectation that the passively transferredRSV antibodies would have inhibited the replication of the g=vaccine,rendering it non-immunogenic and non-protective has, suprisingly, notbeen borne out

TABLE 16 Replication of RSV cpts-248/404, cpts-248, or cpts-530/1009 inthe upper and lower respiratory tract of seronegative chimpanzees, someof which were infused with RSV neutralizing antibodies two days prior toimmunization. Reciprocal serum RSV neutralizing Virus replication Animalinfected antibody titer at Nasopharynx Trachea Rhinorrhea with 10⁴ pfuof time of Chimpanzee Duration Peak titer Duration Peak titer scoreindicated virus immunization number (days) (log₁₀pfu/ml) (days)(log₁₀pfu/ml) Peak Mean cpts-248/404 <10 17 0 <0.7 0 <0.7 0 0   <10 20 9<0.7 0 <0.7 0 0   <10 19 8 1.9 0 <0.7 2 0.3 <10 20 9 2.0 0 <0.7 1 0.2(mean 4.3) (mean 1.3) (mean 0) (mean <0.7) (mean 0.8) (mean 0.1) 142 210 <0.7 0 <0.7 2 0.6 256 22 0 <0.7 0 <0.7 1 0.1 (mean 0) (mean <0.7)(mean 0) (mean <0.7) (mean 1.5) (mean 0.4) cpts-530/1009 <10  1 9 3.1 0<1.0 1 0.3 <10  2 10  4.0 10  1.8 1 1.1 <10  3 9 4.0 0 <1.0 1 0.6 <10  49 3.3 0 <1.0 1 0.5 (mean 9.3) (mean 3.6) (mean 2.5) (mean 1.2 (mean 1.0)(mean 0.6) 259 23 8 3.0 0 <0.7 1 0.1 190 24 7 1.2 0 <0.7 1 0.2 (mean7.5) (mean 2.1) (mean 0) (mean <0.7) (mean 1.0) (mean 0.2) cpts-248 <1025 10  4.6 8 5.4 1 0.2 <10 26 10  4.5 6 2.2 1 0.1 <10 27 9 4.7 10  2.1 10.1 <10 28 9 4.2 8 2.2 1 0.1 (mean 9.5) (mean 4.5) (mean 8.0) (mean 3.0)(mean 1.0) (mean 0.1) 290 29 13  4.2 0 <0.7 2 0.4 213 30 16  4.7 0 <0.73 0.9 (mean 14.5) (mean 4.5) (mean 0) (mean <0.7) (mean 2.5) (mean 0.7)

TABLE 17 Serum antibody response of chimpanzees immunized on day 0 withRSV cpts-248/404, cpts-248, or cpts-530/1009, in the presence or absenceof passively-transferred antibodies, and challenged 4 to 6 weeks laterwith wild-type RSV A2 Serum antibody titer (reciprocal of geometricmean) IgG ELISA RSV F RSV G Neutralizing³ Day 0 Day 0 Day 0 Post- Animal(48 hrs. (48 hrs. (48 hrs. challenge infected No. Infused after Post- 28days after Post- 28 days after Post- 28 days neut./ELISA with of withPrior infusion im- post- Prior infusion im- post- Prior infusion im-post- antibody titer indicated ani- anti- to of anti- muni- chal- to ofanti- muni- chal- to of anti- muni- chal- ratio virus mals bodies studybodies) zation¹ lenge² study bodies) zation¹ lenge² study bodies)zation¹ lenge² F G cpts-248/ 4 no <40   <40 6,400  2,560  60  60 1,0001,600 <10 <10 208   362 0.2 0.1 404 2 yes <40 1,600   640 25,600 100 1001,600 21,760  <10 199 111 92,681 4.3 3.6 cpts-530/ 4 no <40   <40 6,40010,240 <40 <40 10,240  2,560 <10 <10 256   2521 1.0 0.3 1009 2 yes <401,600   640 10,240  40 100   400 10,240  <10 225  52 37,641 3.7 3.7cpts-248 4 no <40   <40 7,840  6,400 <40 <40   250 2,560 <10 <10 147  338 0.1 0.1 2 yes <40   640 1,600  5,400  40  40 1,600 5,440 <10 252119 26,616 4.9 4.9 ¹The day on which postimmunization titer wasdetermined was also the day on which challenge was performed, i.e. day28 for animals not infused with antibody, day 42 for animals infused.²Values determined from samples taken 28 days after challenge. Challengeperformed on day 28 postimmunization for animals not infused withantibody, day 42 for animals infused. ³Determined by complement-enhanced60% plaque reduction of RSV A2 in HEp-2 cell monolayer cultures.Neutralizing antibody titer represents the mean value from two tests.

TABLE 18 Immunization of chimpanzees with RSV cpts-248, cpts-248/404, orcpts-530/1009 induces resistance to wild-type RSV A2 challenge 4-6 weekslater. Passively- Replication of RSV A2 challenge virus^(a) transferredNasopharynx Tracheal lavage Rhinorrhea Virus used for RSV antibodiesChimpanzee Duration Peak titer Duration Peak titer score immunizationpresent number (days) (log₁₀pfu/ml) (days) (log₁₀pfu/ml) Mean^(b) Peakcpts-248/404 no 17^(c) 0 <0.7 0 <0.7 0 0 no 18^(c) 8 3.4 0 <0.7 0 0 yes21  6 2.7 0 <0.7 0.5 2 yes 22  0 <0.7 0 <0.7 0 0 cpts-530/1009 no 1 72.1 0 <0.7 0 0 no 2 0 <0.7 0 <0.7 0 0 yes 23  6 2.5 0 <0.7 0.5 1 yes 24 7 2.0 0 <0.7 0.2 1 cpts-248 no 25^(c) 5 2.7 0 <0.7 0 0 no 26^(c) 9 1.8 0<0.7 0 0 yes 29  0 <0.7 0 <0.7 0 0 yes 30  6 2.4 0 <0.7 1.2 3 none no13^(d) 9 5.1 13  5.4 1.0 1 no 14^(d) 9 6.0 8 6.0 1.7 4 no 15^(c) 13  5.38 5.9 2.1 3 no 16^(c) 9 5.4 8 5.6 1.0 3 ^(a)Animals which were immunizedwith indicated virus 4 to 6 weeks prior were challenged with 10⁴ pfu ofRSV A2 wild-type virus. ^(b)Mean rhinorrhea scores represent the sum ofscores during the eight days of peak virus shedding divided by eight.Four is the highest score; zero is the lowest score and representscomplete absence of detectable rhinorrhea. ^(c)Animals from Crowe et al.Vaccine 12:691-699 (1994). ^(d)Animals from Collins et al. Vaccine8:164-168 (1990).

EXAMPLE II Use of Cold Adaptation Attenuate cpRSV Mutants

This Example describes the introduction of growth restriction mutationsinto incompletely attenuated host range-restricted cpRSV strains byfurther passage of the stains at increasingly reduced temperatures toproduce derivative strains which are more satisfactorily attenuated foruse in human vaccines.

These cold-adaptation (ca) approaches were used to introduce furthersattenuation into the cpRSV 3131 virs, which is incompletely attenuatedin seronegative children.

Under the first strategy, a parent stock of cold-passaged RSV A2 (cpRSV3131) obtained from Flow Laboratories was prepared by passage in MRC-5cells at 25° C. as described in Example I. Briefly, cold-passaged viruswas inoculated into MRC-5 or Vero cell monolayer culture at amultiplicity of infection of ≦0.01 and the infected cells were incubatedfor 3 to 14 days before subsequent passage. Virus was passaged over 20times at 20-22° C. to derive more attenuated virus. The technique ofrapid passage, as soon as the first evidence of virus replication isevident (i.e., 3 to 5 days), was preferable for selection of mutantsable to replicate efficiently at low temperatures. Additionally, an RSVsubgroup B strain, St. Louis/14617/85 clone 1A1, was isolated in primaryAfrican Green monkey kidney cells, passaged and cloned in MRC cells(1A1-MRC14), and cold-passaged 52 times in MRC-5 or Vero cells at 32 to22° C.

A second strategy employed a biologically cloned derivative of theuncloned parental cpRSV 3131 virus. This virus was biologically clonedin bovine embryonic kidney (BEK) cells [the tissue used to originallyderive the cpRSV 3131 virus—see Friedewald et al., J. Amer. Med. Assoc.204:690-694 (1968)]. This cloned virus was then passaged at 10 dayintervals in Vero cells at low temperature. Alternatively, the cpRSV3131 virus was cloned by two serial terminal dilutions (TD2P4) in MRC-5cells and passaged at 10day intervals in MRC-5 or Vero cells.

The third strategy involved selection of mutants that produce largeplaques at low temperature. An RSV cp3131 derivative virus designatedplaque D1 that produces large plaques at 25° C. has been identified.This virus was derived from the third passage (P3) level of thecp3131-17 (BEK) lineage cp3131-17 (BEK) lineage. The largest plaqueproduced by P3 virus was amplified at 32° C., then re-plaqued at 250° C.Once again the largest plaque was selected, amplified, and re-plaqued.After five such cycles, large plaque mutant virus D1 was obtained D1 wasbiologically cloned by two additional cycles of plaque-to-plaquepurification at 25° C.

Biologically cloned virus D1 produces distinctly and uniformly largerplaques at 25° C. than cp3131 or wild type virus A2. Thus D1 is coldadapted by the criterion of large plaque size at 25° C. Efficiency ofplaque formation studies demonstrated that D1 is not temperaturesensitive. At 37° C., D1 plaques are indistinguishable from those ofwild-type RSV or cp3131, suggesting that D1 is not restricted in growthat this temperature. Consistent with this, D1 produces extensivecytopathic effects in Vero cell monolayers at 37° C. and 40° C. (i.e.the highest temperatures tested).

EXAMPLE III Introduction of Further Attenuating Mutations into ts-RSV

This Example describes the use of ts mutants as parental viruses toproduce more completely attenuated strains. Two RSV A2 ts mutants wereselected for this process, namely ts-4 and ts-1 NG1. Two distinctmethods were chosen to introduce additional mutations into the RSV tsmutants. First, the incompletely attenuated RSV ts mutant was subjectedto chemical mutagenesis, and mutagenized progeny that are moretemperature-sensitive with regard to plaque formation were selected forfurther analysis. Second, the RSV ts mutants were passaged at lowtemperature to select RSV ts mutants with the ca phenotype, i.e.,increased capacity to replicate at suboptimal temperature compared towild-type parental virus.

A parent stock of ts-1 NG1 virus was prepared from Flow Laboratories LotM4 of live Respiratory Syncytial Virus (A-2) ts-1 NG-1 mutant, MRC-5grown virus. This mutant, derived from the ts-1 mutant by a second roundof mutagenesis using nitrosoguanidine, possesses two or more independentts mutations, but still induces substantial rhinorrhea in susceptiblechimpanzees. This virus was passaged twice in Vero cells at 32° C. tocreate a ts-1 NG-1 suspension for mutagenesis. The virus was then grownin the presence of 4×10⁻⁴(M5-fluorouracil to induce additional mutationsduring replication or was exposed to 5-azacytidine at 36° C. after5-fluorouracil treatment The mutagenized stock was then analyzed byplaque assay on Vero cells that were maintained under an agar overlay,and, after an appropriate interval of incubation, plaques wereidentified microscopically. 586 plaques were picked, and the progeny ofeach plaque were separately amplified by growth on fresh monolayers ofVero cells. The contents of each of the tissue cultures inoculated withthe progeny of a single plaque of mutagenized ts-1 NG-1 virus wereseparately harvested when cytopathic effects on the Vero cells appearedmaxima Progeny virus that was more temperature-sensitive than ts-1 NG1was sought by titering these plaque pools on HEp-2 cells at 32° C. and36° C. Any virus exhibiting greater temperature sensitivity than ts-1NG1 (i.e., 100-fold or greater reduction in titer at restrictivetemperature [36° C.] compared to 32° C.) was evaluated further. Sixplaque progeny more ts than the RSV ts-1 NG-1 parent virus wereidentified and these strains were biologically cloned by serialplaque-purification on Vero cells three times, then amplified onVero-cells. The cloned strains were titered at 32° C., 35° C., 36° C.,37° C., and 38° C. (efficiency of plaque formation assay) to confirmtheir ts phenotype. Efficiency of plaque formation data generated byassay on HEp-2 cells further confirmed the phenotype of the six mutants(Table 19).

The two most ts viruses, A-20-4 and A-37-8, were highly attenuated inmice compared to their ts-1 NG1 parent virus, indicating thatacquisition of increased level of temperature sensitivity wasaccompanied by augmented attenuation (Table 20). These viruses wereinfectious for mice because they induced an antibody response. The ts-1NG1/A-20-4 virus is attenuated for chimpanzees (Table 21) and infectionof chimpanzees with ts-1 NG1/A-20-4 induced resistance to wild-typevirus challenge (Table 22). Significantly, rhinorrhea does not occur.

Mutagenesis of the ts-4 virus was also performed, using the same methodas for mutagenesis of ts-1 NG1, virus. Mutations were also introducedinto the ts-4 viruses by cold-passage. The ts4 virus replicates to hightiter at 22° C. after 43 cold-passages. Six plaque progeny that weremore ts than the RSV ts-4 parent virus were identified (Table 23). Thets-4 cp-43 is even further restricted in replication in Balb/c mice(Table 24).

TABLE 19 Efficacy of plaque formation of ts-1 NG1 derivatives Titer(log₁₀pfu/ml) at indicated temperature Virus 32° 35° 36° 37° 38°A-20-4(4-1)^(a) 5.9* <1 <1 <1 <1 A-37-8(1-2)^(a) 6.3 6.3 <1 <1 <1 A-15-73.5 ND 2.1 1.5 <1 A-25-8 5.3 ND 5.0* 4.8* <1 A-21 5.1** ND 4.8** 4.5**<1 Ts1NG1 6.6 6.6 6.5 6.6 <1 ^(a)3x plaque purified *Small-plaquephenotype (<50% wild-type plaque size) **Pinpoint-plaque phenotype (<10%wild-type plaque size) ND = Not Done

TABLE 20 Replication of ts-1 NG1 parent and progeny viruses in Balb/cmice Dose Day Post- Titer in lung Titer in nose Virus (log₁₀pfu)Infection 32° 38° 32° 38° A2 wt 6.1 4 4.66 ± 4.80 ± 3.18 ± 3.29 ± 32^(a)0.16 0.40 0.33 5 5.18 ± 5.25 ± 3.40 ± 3.47 ± 0.33 0.23 0.20 0.14 Ts1NG15.8 4 4.31 ± <2.0 2.82 ± <2.0 0.17 0.25 5 3.98 ± <2.0 2.74 ± <2.0 0.120.31 Ts1NG1/ 6.1 4 <2.0 <2.0 <2.0 <2.0 A-20-4 5 <2.0 <2.0 <2.0 <2.0Ts1NG1/ 6.3 4 <2.0 <2.0 <2.0 <2.0 A-37-8 5 <2.0 <2.0 <2.0 <2.0 ^(a)Meanlog₁₀pfu/g of indicated tissue ± standard error. 6 animals/group.

TABLE 21 Replication of ts-1 NG1/A-20-4, ts-1 NG1, ts-1 or wild-type RSVA2 in the upper and lower respiratory tract of seronegative chimpanzees.Virus replication Nasopharynx Trachea Animal infected with Route ofChimpanzee Duration^(b) Peak titer Duration^(b) Peak titer Rhinorrheascores indicated virus Inoculation^(a) number (Days) (log₁₀pfu/ml)(Days) (log₁₀pfu/ml) Mean^(c) Peak ts-1NG1/A-20-4 IN + IT 15  0 <0.7 0<0.7 0 0 IN + IT 16  0 <0.7 0 <0.7 0 0 IN + IT 17  0 <0.7 0 <0.7 0 0IN + IT 18  16^(d) 2.7 0 <0.7 0 0 mean 4.0 mean 1.2 mean 0 mean <0.7mean 0 mean 0 ts-1 NG1 IN 19^(e) 8 4.2 0 <1.1 0.6 1 IN 20^(e) 7 3.9 0<1.1 0.7 1 IN 21^(e) 13  5.4 0 <1.1 0.4 1 IN 22^(e) 10  5.2 10d 3.7d 0.62 mean 9.5 mean 4.7 mean 2.5 mean 1.8 mean 0.6 mean 1.3 ts-1 IN 23^(e)16  3.4 0 <1.1 0.4 1 IN 24^(e) 13  4.4 0 <1.1 1.0 3 IN 25^(e) 13  5.013d 2.2 2.0 4 IN 26^(e) 10  3.4 0 <1.1 1.0 2 mean 13 mean 4.1 mean 3.3eman 1.4 mean 1.1 mean 2.5 A2 wild-type IN  9^(b) 9 5.1 13  5.4 1.0 1 IN10^(b) 9 6.0 8 6.0 1.7 4 IN + IT 11^(e) 13  5.3 8 5.9 2.1 3 IN + IT12^(e) 9 5.4 8 5.6 1.0 3 mean 10 mean 5.5 mean 9.3 mean 5.7 mean 1.4mean 2.8 ^(a)IN = Intranasal only; IN + IT = Both intranasal andintratracheal administration. ^(b)Indicates last day post-infection onwhich virus was recovered. ^(c)Mean rhinorrhea score represents the sumof daily scores for a period of eight days surrounding the peak day ofvirus shedding, divided by eight. Four is the highest score; zero is thelowerst score. ^(d)Virus isolated only on day indicated. ^(e)Animalsfrom Crowe, et al., Vaccine 11:1395-1404 (1993).

TABLE 22 Immunization of chimpanzees with 10⁴ pfu of RSV ts-1NG1/A-20-4, ts-1 NG1, or ts-1 induces resistance to 10⁴ pfu RSV A2wild-type virus challenge on day 28. Serum Virus Recovery neutralizingantibody Nasopharynx Trachea titer (reciprocal log₂) on Virus used toChimpanzee Duration Peak titer Duration Peak titer Rhinorrhea scores dayindicated^(d) immunize animal number (Days) (log₁₀pfu/ml) (Days)(log₁₀pfu/ml) Mean^(a) Peak Day 28 Day 49 or 56 ts-1 NG1/A-20-4 15  0<0.7 0 <0.7 0 0 <3.3 10.7 16  0 <0.7 0 <0.7 0 0 <3.3 11.9 17  0 <0.7 0<0.7 0 0 5.3 10.3 18  3 2.0 0 <0.7 0 0 8.2 11.8 mean 0.8 mean 1.0 mean 0mean <0.7 mean 0 mean 0 mean 5.0 mean 11.2 ts-1 NG1 19^(b) 0 <0.7 0 <1.10 0 11.1 9.8 20^(b) 0 <0.7 0 <1.1 0 0 12.7 9.1 21^(b) 0 <0.7 0 <1.1 0 010.8 11.0 22^(b) 0 <0.7 0 <1.1 0 0 10.0 8.6 mean 0 mean <0.7 mean 0 mean<1.1 mean 0 mean 0 mean 11.1 mean 9.6 ts-1 23^(b) 0 <0.7 0 <1.1 0 0 9.410.5 29^(b) 0 <0.7 0 <1.1 0 0 12.4 12.8 25^(b) 5 0.7 0 <1.1 0 0 9.0 9.626^(b) 5 0.7 0 <1.1 0 0 13.4 12.0 mean 2.5 mean 0.7 mean 0 mean <1.1mean 0 mean 0 mean 11.0 mean 11.2 none  9^(c) 9 5.1 13  5.4 1.0 1 <3.311.0 10^(c) 9 6.0 8 6.0 1.7 4 <3.3 9.8 11^(b) 13  5.3 8 5.9 2.1 3 <3.39.4 12^(b) 9 5.4 8 5.6 1.0 3 <3.3 14.5 mean 10 mean 5.5 mean 9.3 mean5.7 mean 1.5 mean 2.8 mean <3.3 mean 11.1 ^(a)Mean rhinorrhea scorerepresents the sum of scores during the eight days of peak virusshedding divided by eight. Four is the highest score; zero is the lowestscore. ^(b)Animals from Crowe, et al., Vaccine 11:1395-1404 (1993).^(c)Animals from Collins, et al., Vaccine 8:164-168 (1990). ^(d)Serumneutralizing titers in this table were determined in a new assaysimultaneously with other specimens represented in the table.

TABLE 23 The efficiency of plaque formation of six mutants derived fromRSV ts-4 and tested in HEp-2 cells at permissive and restrictivetemperatures, compared with controls The titer of virus (log₁₀pfu/ml)Small- Shut-off at the indicated temperature (° C.) plaques temperatureVirus 32 33 34 35 36 37 38 39 40 at 32° C. (° C.)¹ A2 wild-type 5.7 5.85.5 5.5 5.3 5.5 5.5 5.4 5.5 no >40  ts-4 4.5 4.7 4.4 4.7 4.7 4.1 3.7 3.02.5 no 40 ts-4 cp-43 6.2 6.1 6.1 6.0 4.4* 4.2** 1.7** 0.7** <0.7 no 37ts-4/20.7.1 6.0 5.9 5.7 5.7* 4.5** 1.8 <0.7 <0.7 <0.7 no 37 ts-4/19.1.25.8 5.7 5.5 5.6* 4.4** <0.7 <0.7 <0.7 <0.7 no 37 ts-4/15.8.2 5.3* 5.4*4.8* 4.9* 2.8** <0.7 <0.7 <0.7 <0.7 yes 36 ts-4/29.7.4 5.7 5.6 5.6 5.7*<0.7 <0.7 <0.7 <0.7 <0.7 no 36 ts-4/31.2.4 4.7 4.2 4.1 4.0* <0.7 <0.7<0.7 <0.7 <0.7 no 36 ¹Shut-off temperature is defined as the lowestrestrictive temperature at which a 100-fold or greater reduction ofplaque titer is observed (bold figures in table). *Small-plaquephenotype (<50% wild-type plaque size) **Pinpoint-plaque phenotype (<10%wild-type plaque size)

TABLE 24 Replication of RSV ts-4 and RSV ts-4 cp-43 in Balb/c mice¹Shutoff Virus titer (mean log₁₀pfu/g tissue Virus used to temperature ofof six animals ± standard error) infect animals: virus (° C.) Nasalturbinates Lungs A2 wild-type >40 5.0 ± 0.14 5.2 ± 0.05 ts-4 39 4.3 ±0.09 4.7 ± 0.11 ts-4 cp-43 37 2.1 ± 0.09 2.7 ± 0.27 ¹Mice wereadministered 10^(6.3) p.f.u. intranasally under light anesthesia on day0, then sacrificed by CO₂ asphyxiation on day 4.

EXAMPLE IV RSV Subgroup B Vaccine Candidates

This Example describes the development of RSV subgroup B virus vaccinecandidates. The same approach used for the development of the subgroup Amutants of the invention was utilized for the subgroup B viruses. Aparent stock of wild-type B-1 RS virus was cold-passaged 52 times inVero cells at low temperature (20-25° C.) and the virus was subjected toplaque, purification at passages 19 and 52. Three of the clones derivedfrom the passage 52 suspension were evaluated independently, and oneclone, designated RSV B-1cp52/2B5, was selected for further evaluationbecause it was highly attenuated in the upper and lower respiratorytract of the cotton rat (Table 25). An evaluation of several clones atdifferent passage levels of the cpRSV B-1 virus indicate that the RSVB-1cp52/2B5 mutant sustained three mutations that independentlycontribute to its attenuation phenotype. The RSV B-1cp52/2B5 mutantretained its attenuation phenotype following prolonged replication inimmunosuppressed cotton rats (Table 26). This finding of a high level ofgenetic stability is consistant with the fact that it possesses threemutations contributing to the attenuation phenotype.

Further evaluation of the subgroup B mutants in order to characterizethem in a similar manner as the subgroup A mutants, was carried out inCaribbean Green monkeys (Tables 27 and 28) and chimpanzees (Table 29).Monkeys immunized with either RSV B-1 cp-23 or cp-52/2B5 were resistantto replication of RSV B-1 wild-type virus, indicating that infectionwith the highly attenuated derivatives of the RSV B-1 wild-type viruswas sufficient to induce resistance to wild-type challenge (Table 27).The results in the seronegative chimpanzee, like that in the Greenmonkeys, clearly evidence the attenuation of the RSV B-1cp52/2B5 in theupper and lower respiratory tracts.

The RSV B-1 cp52/2B5 mutant has been further mutagenized with5-fluorouracil and the resulting plaques picked and screened at 32° vs.38° C. for the ts phenotype. The selected cpts mutants wereplaque-purified three times in Vero cells and then amplified twice inVero cells. As a result, seven cpts mutants of RSV B-1cp52/2B5 have beenidentified (Table 30) and their level of replication in cotton rats hasbeen studied (Table 31). One of these mutants, namely cpts176, wasfurther mutagenized and a series of mutant derivatives were obtainedthat were more a in than the RSV B-1 cpts176 parent virus (Table 32).

As with the subgroup A mutants of the invention, the subgroup B mutantsare infectious and exhibit a significant degree of attenuation forcotton rats, monkeys, and chimpanzees. Despite attenuation in vivo, theRSV B-1 cp mutant viruses induced resistance in monkeys againstwild-prechallenge. The ts mutants of the RSV B-1 cp52/2B5 virus areattenuated and demonstrate a more restricted level of replication in thenasopharynx and lungs of the cotton rat than the RSV B-1 cp52/2B5 parentvirus.

TABLE 25 Replication in cotton rats of RSV B-1 wild-type compared withfive plaque-purified cold-passaged mutants derived from RSV B-1, in twoseparate experiments Virus recovery (log₁₀pfu/g tissue) on day 4* Virusused to infect Nasal turbinates Lungs animals on day 0** Exp. 1 Exp. 2Exp. 1 Exp. 2 RSV B-1 wild-type 4.7 ± 0.14 5.1 ± 0.10 5.4 ± 0.15 5.8 ±0.08 RSV B-1 cp-12/B1A nd 3.3 ± 0.15 nd 4.4 ± 0.10 RSV B-1 cp-23/1A1 nd2.4 ± 0.36 nd 3.2 ± 0.31 RSV B-1 cpsp-52/1A1 1.7 ± 0.11 2.1 ± 0.27 3.0 ±0.13 2.3 ± 0.07 RSV B-1 cp-52/2B5 1.8 ± 0.25 2.2 ± 0.3  1.8 ± 0.11 <1.5RSV B-1 cp-52/3C1 1.8 ± 0.14 nd 1.8 ± 0.14 nd RSV A2 5.9 ± 0.09 5.4 ±0.07 6.6 ± 0.06 6.1 ± 0.06 RSV A2 cpts-530/1009 3.2 ± 0.11 2.1 ± 0.222.1 ± 0.19 1.7 ± 0.12 *Virus recovery determined by titration of tissuehomogenates on Vero cell monolayer cultures at 32° C. with a 10-dayincubation in Experiment 1, 7-day incubation in Experiment 2. **Cottonrats infected intranasally with 10^(5.5) pfu of indicated virus. nd =not done

TABLE 26 Growth in cotton rats of day 14 isolates* from RSV B-1cp52/2B5-infected immunosuppressed cotton rats compared with controlsVirus recovery Reduction of replication versus Virus titer on day 4 inindicated tissue RSV B-1 wild-type Virus (mean log₁₀pfu/g tissue ±standard (log₁₀pfu/g) infected error of the mean) Nasal animals^(a)Nasal turbinates^(b) Lungs^(c) turbinates Lungs RSV B-1 3.9 ± 0.03 (6/6)4.8 ± 0.12 (6/6) — — wild-type RSV B-1 2.0 ± 0.07 (8/8) <1.5 (0/8)1.9 >3.3 cp 52/2B5 isolate 1 1.5 ± 0.13 (5/8) 1.5 ± 0.04 (1/8) 2.5 3.3isolate 2 1.5 ± 0.13 (6/8) <1.5 (0.8) 2.4 >3.3 isolate 3 1.5 ± 0.16(3/8) <1.5 (0/8) 2.5 >3.3 isolate 4 1.3 ± 0.09 (4/8) <1.5 (0/8) 2.6 >3.3isolate 5 1.2 ± 0.00 (2/8) <1.5 (0/8) 2.7 >3.3 isolate 6 1.2 ± 0.00(3/8) <1.5 (0/8) 2.7 >3.3 isolate 7 1.3 ± 0.06 (3/8) <1.5 (0/8) 2.7 >3.3*Isolates were virus suspensions obtained following amplification by oneVero cell tissue culture passage of virus present in the original nasalturbinate homogenate on day 14 of an immunosuppressed cotton rat.^(a)Groups of 8 cotton rats infected with 10^(5.5) pfu of indicatedvirus in a 0.1 ml inoculum on day 0. ^(b)( ) indicates the numbers ofanimals from which virus was detected at 1.2 log₁₀pfu/g or greater.^(c)( ) indicates the numbers of animals from which virus was detectedat 1.5 log₁₀pfu/g or greater.

TABLE 27 Replication in Caribbean Green monkeys of RSV A2 and RSV B-1wild- types compared with that of two cold-passaged mutants derived fromRSV B-1, followed by homologous or heterologous RSV A2 or B-1 wild-typechallenge Challenge Immunization Tracheal Virus used to NP swab Tracheallavage NP swab lavage infect animals Peak Days Peak Days Challenge PeakPeak on day 0^(a) titer^(b) shed^(b) titer shed virus titer^(b)titer^(b) A2 3.4 9 <0.7 0 A2 <0.7 <0.7 3.5 7 <0.7 0 A2 <0.7 <0.7 3.5 94.8 10  A2 <0.7 <0.7 3.2 8 0.7 7 A2 <0.7 <0.7 1.7 6 <0.7 0 B-1 <0.7 <0.73.5 10  <0.7 0 B-1 <0.7 <0.7 2.4 8 0.7 0 B-1 <0.7 <0.7 4.2 9 <0.7 0 B-1<0.7 <0.7 mean 3.2 mean 8.3 mean 1.2 B-1 2.8 9 1.5 10* B-1 <0.7 <0.7 2.39 1.9 7 B-1 <0.7 <0.7 2.2 7 1.7 10* B-1 <0.7 <0.7 2.2 9 1.3 10* B-1 <0.7<0.7 1.6  8* 1.2  5* A2 <0.7 <0.7 2.1 10  1.7  7* A2 <0.7 <0.7 mean 2.2mean 8.7 mean 1.6 mean <0.7 mean <0.7 B-1 cp-23 1.8 14  <0.7 0 B-1 <0.7<0.7 1.3 5 <0.7 0 B-1 <0.7 <0.7 2.0 8 0.7 10  B-1 <0.7 <0.7 1.7 4 <0.7 0B-1 <0.7 <0.7 mean 1.7 mean 7.8 mean <0.7 mean <0.7 mean <0.7 B-1 cp-521.3 8 <0.7 0 B-1 <0.7 <0.7 1.3 4 <0.7 0 B-1 <0.7 <0.7 1.3 7 <0.7 0 B-1<0.7 <0.7 0.7  3* <0.7 0 B-1 <0.7 <0.7 mean 1.2 mean 5.5 mean <0.7 mean<0.7 mean <0.7 ^(a)Animals infected intratracheally and intranasallywith 10^(5.5) p.f.u. virus at each site in a 1.0 ml inoculum on day 0.^(b)Log₁₀pfu/ml titers determined by plaque assay on HEp-2 cellmonolayer cultures for RSV A2, and Vero cell monolayer cultures for RSVB-1 and its derivatives. *Virus detected only on day indicated.

TABLE 28 Neutralizing antibody response of Caribbean Green Monkeysinfected with RSV A2, RSV B-1, or B-1 cp derivatives, then challengedwith homologous or heterologous wild-type virus one month later. Animalsinfected on Day 28 60% Plaque reduction serum neutralizing titer againstindicated virus (reciprocal mean) day 0 with indicated challenge virusRSV A2 RSV B-1 virus (number of (number of Post-infection Post-challengePost-infection Post-challenge animals) animals) Day 0 (day 28) (day 56)Day 0 (day 28) (day 56) A2 (8) A2 (4)  <10 53,232 40,342 <10 1,552 1,911B-1 (4) 23,170 1,911 B-1 (6) B-1 (4) <10 3,327  3,822 <10 2,048 2,521 A2(2)  30,574 35,120  B-1 cp-23 (4) B-1 (4) <10 6,208 10,086 <10 4,7057,132 B-1 cp-52/2B5 (4) B-1 (4) <10   194 16,384 <10   239 3,822Antigenic relatedness of RSV A2 and RSV B-1 by cross-neutralization inGreen Monkeys, calculated using Archetti-Horsfall formula = 47%$R = {100 \times \frac{\left( {{heterologous}{\quad \quad}2} \right)}{\left( {{homologous}{\quad \quad}1} \right)} \times \frac{\left( {{heterologous}{\quad \quad}1} \right)}{\left( {{homologous}{\quad \quad}2} \right)}}$

TABLE 29 The replication of RSV B-1 or RSV B-1 cp-52 in seronegativechimpanzees following simultaneous intratracheal and intranasaladministration. Virus replication Animal infected with InfectionNasopharynx Trachea indicated virus dose Duration^(b) Peak titerDuration^(b) Peak titer Rhinorrhea score on day 0 (pfu) Exp. (days)(log₁₀pfu/ml) (days) (log₁₀pfu/ml) Peak Mean^(c) RSV B-1 wild-type 10⁴ 19 3.7 8 3.2 1 0.5 1 10  3.5 0 <0.7 2 0.9 1 10  2.8 0 <0.7 3 1.1 1 10 2.7 8 3.4 3 0.9 avg. 9.8 mean 3.2 avg. 4.0 mean 2.0 mean 2.3 mean 0.910⁵ 2 7 2.8 8 1.0 1 1.0 2 7 3.3 4 3.9 3 1.3 avg. 7.5 mean 3.1 avg. 6.0mean 2.5 mean 2.0 mean 1.1 B-1 cp-52/2B5 10⁴ 1 5 1.5 0 <0.7 0 0 1 0 <0.70 <0.7 0 0 10⁵ 3 0 <0.7 0 <0.7 0 0 3 0 <0.7 0 <0.7 0 0 avg 1.2 mean 0.9avg. 0 mean <0.7 mean 0 mean 0 ^(a)These data were combined from threeseparate experiments, the infection dose of indicated virus in the firstexperiment was 10⁴, the second and third experiments were 10⁵.^(b)Indicates the last day post-infection on which virus was recovered.^(c)Mean rhinorrhea score represents the sum of daily scores for aperiod of eight days surrounding the peak day of virus shedding, dividedby eight. Four is the highest score; zero is the lowest score.

TABLE 30 The efficiency of plaque formation of eight mutants derivedfrom RSV B-1 cp52/2B5 Plaque titer (log₁₀pfu/ml in Vero of HEp-2 Cellsat indicated temperature (° C.) HEp-2 Vero HEp-2 Shutoff RSV 32 32 35 3637 38 39 temp (° C.) B-2 wild-type 6.1 5.8 5.7 5.6 5.6 5.7 5.5 >39  B-1cp-52/2B5 5.9 5.4 5.2 5.1 5.0 5.0 4.7** >39  cpts-452 6.1 5.6 5.2 5.23.3** 3.1** <0.7 37 cpts-1229 5.7 5.1 4.9 5.1 4.4** <0.7 <0.7 38cpts-1091 5.7 5.1* 4.7** 5.2** <0.7 <0.7 <0.7 37 cpts-784 5.1 4.3* 4.0**4.1** <0.7 <0.7 <0.7 37 cpts-176 6.1 5.4* 4.8* 5.0** <0.7 <0.7 <0.7 37cptssp-1415^(a) 5.8 <0.7 <0.7 <0.7 <0.7 <0.7 <0.7 38 cpts-1324 5.9 5.15.0* 5.0 <0.7 <0.7 <0.7 37 cpts-1313 5.7 3.9** 3.0** <0.7 <0.7 <0.7 <0.736 A2 6.4 6.3 6.3 6.3 6.3 6.3 6.3 >39  A2/248 6.3 6.3 6.2 6.3 5.8 <0.7<0.7 38 A2/248/404 4.4 4.3 3.3 4.0 <0.7 <0.7 <0.7 37 A2/248/955 4.8 4.84.8 4.4 <0.7 <0.7 <0.7 37 *Small-plaque phenotype (<50% wild-type plaquesize). **Pinpoint-plaque phenotype (<10% wild-type plaque size). Boldfigures denote shutoff temperatures (defined as the lowest restrictivetemperature at which a 100-fold or greater reduction of plaque titer wasobserved). ^(a)At 32° C., no plaques were observed. Therefore, noshut-off temperature was determined by efficiency of plaque formation.The mutant was assigned a shutoff temperature of 38° C., in HEp-2 cellculture as determined by a 100-fold decrease in virus yield (TCID₅₀) inliquid medium overlay.

TABLE 31 Level of replication in cotton rats of seven ts mutants derivedfrom RSV B-1 cp-52/2B5 Replication in cotton rats¹ (mean log₁₀pfu/gtissue of six animals ± s.e.) RSV Nasal turbinates Lungs B-1 wild-type4.3 ± 0.05 (6/6)² 4.4 ± 0.25 (6/6) B-1 cp-52/2B5 1.7 ± 0.11 (6/6) <1.5(0/6) cpts-452 1.4 ± 0.1 (3/6) <1.5 (0/6) cpts-1091 1.7 ± 0.07 (4/6)<1.5 (0/6) cpts-784 1.5 (1/6) <1.5 (0/6) cpts-1229 1.4 ± 0.15 (3/6) <1.5(0/6) cpts-176 1.5 ± 0.17 (3/6) <1.5 (0/6) cptssp-1415 <1.2 (0/6) <1.5(0/6) cpts-1324 <1.2 (0/6) <1.5 (0/6) ¹Cotton rats were inoculatedintranasally with 4.5-5.8 log₁₀pfu under light anesthesia on day 0, thensacrificed by CO₂ asphyxiation on day 4. ²Titer from samples containingvirus only. Parentheses indicate fraction of samples containing virus.

TABLE 32 The efficiency of plaque formation of 14 mutants derived fromRSV B-1 cpts-176, compared with controls In vitro efficiency of plaqueformation in HEp-2 cell monolayer culture The titer of virus(log₁₀pfu/ml) at Shut-off the indicated temperature (° C.) temperatureRSV 32 35 36 37 (° C.)¹ B-1 wild-type 5.6 5.5 5.4 5.3 >39   B-1cp-52/2B5 5.7 5.7 5.6 5.3 >39   B-1 cpts176 5.5 3.5 3.0 1.9 36/37176/645 3.8 3.0 2.6** <0.7 37 176/860 3.1 2.5 2.4 <0.7 37 176/196 3.32.5 2.0** <0.7 37 176/219 2.6 2.3 2.0** <0.7 37 176/18 4.0 3.2 <0.7 <0.736 176/73 2.6 2.0 <0.7 <0.7 36 176/1072 3.2 2.3 <0.7 <0.7 36 176/10382.8 2.2 <0.7 <0.7 36 176/81 2.2 2.0 <0.7 <0.7 36 176/1040 3.2 2.0 <0.7<0.7 36 176/1045 2.5 1.9 <0.7 <0.7 36 176/517 3.1 2.0** <0.7 <0.7 36176/273 2.3 <0.7 <0.7 <0.7 35 176/427 3.5 <0.7 <0.7 <0.7 35**Pinpoint-plaque phenotype (<10% wild-type plaque size) ¹Shut-offtemperature is defined as the lowest restrictive temperature at which a100-fold or greater reduction of plaque titer is observed (bold figuresin table).

EXAMPLE V Bivalent RSV Subgroup A and B Vaccine

Studies with subgroup A and B viruses demonstrate that in vitro, nointerference occurs between wild-type A2 and B-1 viruses, nor betweencpts RSV 530/1009 and RSV B-1 cp-52/2B25 derivatives in Vero cellmonolayer cultures. The in vivo results of bivalent infection in cottonrats are presented in Table 33. These results confirm the in vitroresults, which show no interference between A-2 and B-1 wild-type RSV,and cpts RSV 530/1009 and RSV B-1 cp-52/2B5. It is expected, therefore,that each vaccine virus will induce homotypic immunity against wild-typevirus, since each component of the bivalent vaccine replicates to alevel in the dual infection comparable to that seen during singleinfection. Each virus alone is capable of inducing homotypic resistanceagainst RSV wild-type challenge.

TABLE 33 Bivalent infection of cotton rats with RSV A2 and RSV B-1viruses or mutant derivatives indicates no in vivo interference Virusrecovery from indicated tissue (log₁₀pfu/g) Nasal turbinates LungsViruses used to RSV A RSV A infect animals* titer RSV B titer titer RSVB titer A2 5.4 ± 0.08 — 5.8 ± 0.07 — B-1 — 4.6 ± 0.03 — 5.4 ± 0.12 A2 +B-1 5.2 ± 0.11 3.6 ± 0.07 5.7 ± 0.08 5.0 ± 0.05 A2 cpts-530/1009 3.2 ±0.09 — 1.9 ± 0.15 — B-1 cp-52 — 2.4 ± 0.08 — <1.5 A2 cpts-530/1009 + 2.8± 0.13 2.0 ± 0.14 1.8 ± 0.08 <1.5 B1 cp-52 *Groups of six animalsinfected with 10⁵ pfu intranasally on day 0 in a 0.1 ml inoculum.

Human Studies

The attenuated virus of the invention is administered to human subjectsaccording to well established human RS vaccine protocols, as describedin, e.g., Wright et al., Infect. Immun. 37:397-400 (1982), Kim et al.,Pediatrics 52:56-63 (1973), and Wright et al, J. Pediatr. 88:931-936(1976), which are incorporated herein by reference. Briefly, adults orchildren are inoculated intranasally via droplet with 10³ to 10⁶ PFU ofattenuated virus per ml in a volume of 0.5 ml. Antibody response isevaluated by complement fixation, plaque neutralization, and/orenzyme-linked immunosorbent assay. Individuals are monitored for signsand symptoms of upper respiratory illness. As with administration tochimpanzees, the attenuated virus of the vaccine grows in thenasopharynx of vaccinees at levels approximately 10fold or more lowert&an wild-type virus, and approximately 10fold or more lower whencompared to levels of cpRSV or other incompletely attenuated parentalstrain. Subsequent immunizations are administered periodically to theindividuals as necessary to maintain sufficient levels of protectiveimmunity.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration and understanding, various modifications may be madewithout deviating from the spirit and scope of the invention.Accordingly, the invention is not limited except as by the appendedclaims.

What is claimed is:
 1. An immunogenic composition comprising, in aphysiologically acceptable carrier, at least one attenuated cpRSV 3131D1 mutant respiratory syncytial virus (RSV).
 2. The immunogeniccomposition according to claim 1, which induces an RSV-specific immuneresponse in seropositive and seronegative individuals.
 3. Theimmunogenic composition according to claim 2, wherein said seropositiveindividuals are infants possessing transplacental acquired RSVneutralizing antibodies.
 4. The immunogenic composition according toclaim 1, which further comprises an adjuvant to enhance the immuneresponse.
 5. The immunogenic composition of claim 1, formulated in adose of 10³ to 10⁶ PFU of attenuated virus.
 6. A method for stimulatingRSV-specific immune responses, which comprises administering to anindividual an immunologically sufficient amount of at least oneattenuated cpRSV 3131 D1 mutant respiratory syncytial virus (RSV), in aphysiologically acceptable carrier.
 7. The method of claim 6, whereinthe attenuated virus is administered to the individual in an amount of10³ to 10⁶ PFU.
 8. The method of claim 6, wherein the attenuated virusis administered to the upper respiratory tract of said individual. 9.The method of claim 8, wherein the attenuated virus is administered tothe nasopharynx.
 10. The method of claim 8, wherein the attenuated virusis administered by spray, droplet, or aerosol.
 11. The method of claim6, wherein the attenuated virus is administered to an individualseronegative for antibodies to said virus.
 12. The method of claim 6,wherein the attenuated virus is administered to an individualseropositive to said virus.